Getting into the guts of a salty problem:

Poor animal production from saltbush pastures is due to

inefficient rumen fermentation

Dianne Mayberry

B.Sc. (Agriculture, Hons I)

This thesis is presented for the degree of Doctor of Philosophy of

The University of Western Australia

Faculty of Natural and Agricultural Sciences

School of Animal Biology

2008

i

Summary

The main hypothesis tested in this thesis was that poor animal production from saltbush

pastures is due to the negative effects of high sodium chloride (NaCl) and potassium

chloride (KCl) on the ruminal environment, and subsequent effects on microbial

populations and products of rumen fermentation.

This main hypothesis was tested in two experiments. In the first experiment (Chapter

Four) the effects of saltbush and a formulated high-salt diet on the ruminal environment

and microbial populations were measured over 24-hours following feeding. Feeding

both the saltbush and high-salt diet increased the salinity of the rumen fluid, but the

formulated high-salt diet caused a decrease in ruminal pH while the saltbush caused an

increase. This resulted in differences in the composition of the ruminal microbial

populations between the sheep fed different diets.

In the second experiment (Chapter Five) the effects of saltbush and a formulated high-

salt diet on rumen fermentation were measured. Sheep fed saltbush had inefficient

rumen fermentation and this was only partially explained by the high salt content of the

diet. Diets containing high levels of NaCl and KCl provided low levels of net energy to

sheep, but sheep fed saltbush lost more energy as methane and faecal energy compared

to sheep fed the formulated high-salt diet. Inefficient rumen fermentation could help to

explain poor animal production from saltbush pastures.

Energy supplements such as barley grain can improve the value of saltbush pastures as

feed for sheep, but there is no information on how much supplement is required. A

third experiment (Chapter Six) was designed to test the hypothesis that there would be

an optimal amount of barley required to improve the efficiency of rumen fermentation

in sheep fed saltbush. Barley and straw were combined in a pellet and substituted for

saltbush at 0, 20, 40, 60, 80 and 100% of the maintenance ration. Feeding barley and

straw improved the efficiency of rumen fermentation in sheep fed saltbush, with an

optimal level of supplementation at 60% of the maintenance diet. This is likely to be

lower (approximately 20% of maintenance) if barley is fed without straw.

ii

Table of contents

Summary ………………………………………………………………………….. i

Table of contents …………………………………………………………………. ii

Statement of contribution ………………………………………………………… vi

Publications arising from this thesis ……………………………………………… vii

Acknowledgements ………………………………………………………………. viii

Chapter 1: General introduction ………………………………………………. 1

Chapter 2: Review of the literature ……………………………………………. 3

2.1 Introduction ……………………………………………………………….. 3

2.2 Saltbush …………………………………………………………………… 3

2.3 Animal production from saltbush …………………………………………. 6

2.3.1 Nutritive value ………………………………………………...… 6

2.3.2 Feed intake …………………………………………………...….. 8

2.3.3 Water intake …………………………………………………..… 11

2.3.4 Liveweight gain ……………………………………………..….. 12

2.3.5 Energy balance ……………………………………………..…… 13

2.4 Effect of saltbush and salt on the rumen …………………………………... 16

2.4.1 Physiology …………………………………………………...….. 17

Saliva ………………………………………………………….… 17

Digestion and absorption ………………………………..........… 18

Rumen motility …………………………………………………... 19

2.4.2 Ruminal microbial environment ……………………………...…. 20

pH …………………………………………………………...…... 20

Salinity ………………………………………………………...… 21

2.4.3 Ruminal microbe populations ………………………………..…. 23

2.4.4 Products of microbial fermentation …………………………….. 24

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Volatile fatty acids ………………………………………………. 25

Ammonia ………………………………………………………… 27

Methane …………………………………………………………. 28

2.5 Opportunities for new techniques in rumen microbiology ………………... 29

2.6 Plant secondary compounds ………………………………………………. 31

2.7 Use of supplements to improve animal production from saltbush ………... 32

2.8 Summary …………………………………………………………………... 34

Chapter 3: General materials and methods …………………………………… 38

3.1 Experimental design ………………………………………………………. 38

3.2 Animals …………………………………………………………………… 38

3.3 Diets ……………………………………………………………………….. 39

3.3.1 Feed analysis ……………………………………………………. 40

3.3.2 Digestibility ……………………………………………………... 41

3.4 Rumen samples ……………………………………………………………. 41

3.4.1 Rumen pH and salinity ………………………………………….. 42

3.4.2 Volatile fatty acids ………………………………………………. 42

3.4.3 Molecular analysis ………………………………………………. 42

Chapter 4: Saltbush increases the pH and salinity of the rumen microbial

environment ……………………………………………………………………... 43

4.1 Introduction ……………………………………………………………….. 43

4.2 Materials and methods ……………………………………………………. 44

4.2.1 Experimental design …………………………………………….. 44

4.2.2 Establishment of a rumen cannula ………………………………. 44

4.2.3 Diets ……………………………………………………………... 44

4.2.4 Rumen fluid collection ………………………………………….. 45

4.2.5 Concentration of Na and K ions in rumen fluid ………………… 46

iv

4.2.6 Analysis of microbial populations ………………………………. 46

4.2.7 In vitro experiment ……………………………………………… 48

4.2.8 Statistical analysis ………………………………………………. 48

4.3 Results …………………………………………………………………….. 48

4.3.1 Rumen pH ………………………………………………………. 48

4.3.2 Rumen salinity …………………………………………………... 50

4.3.3 Na concentration ………………………………………………… 50

4.3.4 K concentration …………………………………………………. 51

4.3.5 Microbial populations …………………………………………… 52

4.4 Discussion …………………………………………………………………. 53

Chapter 5: Saltbush decreases the efficiency of rumen fermentation ……….. 59

5.1 Introduction ……………………………………………………………….. 59

5.2 Materials and methods …………………………………………………….. 60

5.2.1 Experimental design …………………………………………….. 60

5.2.2 Diets ……………………………………………………………... 61

5.2.3 Methane production ……………………………………………... 62

5.2.4 Rumen pH, salinity and volatile fatty acid concentration ……….. 62

5.2.5 Enumeration of methanogens …………………………………… 62

5.2.6 Digestibility ……………………………………………………... 63

5.2.7 Statistical analysis ………………………………………………. 63

5.3 Results …………………………………………………………………….. 63

5.3.1 Methane production ……………………………………………... 63

5.3.2 Methanogens …………………………………………………….. 65

5.3.3 Volatile fatty acid concentration ………………………………… 65

5.3.4 Rumen pH and salinity ………………………………………….. 66

5.3.5 Digestibility ……………………………………………………... 66

5.4 Discussion ………………………………………………………………… 67

v

Chapter 6: What is the optimal level of barley to feed sheep grazing

saltbush? …………………………………………………………………………. 75

6.1 Introduction ……………………………………………………………….. 75

6.2 Materials and methods ……………………………………………………. 76

6.2.1 Experimental design …………………………………………….. 76

6.2.2 Diets ……………………………………………………………... 76

6.2.3 Digestibility ……………………………………………………... 78

6.2.4 Rumen pH, salinity and volatile fatty acid concentration ……..… 78

6.2.5 Methane production ……………………………………….…….. 78

6.3 Results …………………………………………………………………….. 79

6.3.1 Rumen pH and salinity ………………………………………….. 79

6.3.2 Digestibility ………………………………………………….….. 80

6.3.3 Methane production ……………………………………………... 80

6.3.4 Volatile fatty acid concentration ………………………………... 80

6.4 Discussion …………………………………………………………………. 82

Chapter 7: General discussion …………………………………………………. 88

References ……………………………………………………………………….. 91

vi

Statement of contribution

The work presented in this thesis is the original work of the author. The experimental

work and manuscript preparation was carried out by myself after discussions with my

supervisors, Dr Philip Vercoe and Dr David Masters.

Dianne Mayberry

July 2008

vii

Publications arising from this thesis

Mayberry, D.E. Masters, D.G. and Vercoe, P.E. (2008) “Grazing saltbush may cause

mineral deficiencies” in Animal Production in Australia: Proceedings of the 27th

Biennial Conference of the Australian Society of Animal Production, 27: 19

Mayberry, D.E. Masters, D.G. and Vercoe, P.E. (2008) “What is the optimal level of

barley to feed sheep eating saltbush?” in Proceedings of the 2nd International Salinity

Forum, CD only

Mayberry, D.E. Masters, D.G. and Vercoe, P.E. (2007) “Saltbush (Atriplex

nummularia) reduces efficiency of rumen fermentation in sheep” Proceedings of the

12th Seminar of the FAO-CIHEAM Sub-Network on Sheep & Goat Nutrition, in press

with Options Méditerranéennes

Mayberry, D.E. Vercoe, P.E. and Masters, D.G. (2007) “The effect of salt and

saltbush on rumen salinity” in Proceedings of the 7th International Symposium on the

Nutrition of Herbivores, p 560

Mayberry, D.E. Masters, D.G. and Vercoe, P.E. (2006) “Saltbush increases methane

production” in 26th Biennial Conference, Australian Society of Animal Production,

Working Papers, short communication number 11

viii

Acknowledgements

Like many people, I started my PhD because I was not ready to face the “real world”

and get a “real job”. In the last four years I have developed a passion for science and

agricultural research, and had many great opportunities that I don’t think I would have

got in the “real world”. Feedback from producers and other people involved in the

industry has made me feel like I might even be able to make a difference!

There are of course many people that I could not have done this without….

First of all, a massive thank you to my supervisors, Dr Phil Vercoe and Dr Dave

Masters. For the never-ending advice, support and encouragement – it has been a real

privilege working with you. Thank you for coming saltbush picking, sampling rumen

fluid in the middle of the night, sticking it out through the disastrous Friday the 13th

sampling, reading through multiple versions of this thesis and for the fabulous

references (not to mention the Greek dancing!).

Thanks to my trusty team of surgeons – Ian Williams, John Beasley, Peter Hutton and

Chris Mayberry – let’s never do that again!

Thanks to Dr Andre Denis-Wright and Andrew Toovey at CSIRO Livestock Industries

for help with the real-time PCR. It was meant to take two days, but you put up with me

for almost six weeks until it finally worked! Also Dr Yvette Williams for help with

using the methane chambers.

Thanks to Michael Smirk of Soil Science at UWA for letting me put rumen fluid

through your precious AAS (and guiding me through the process).

To all the staff and students at UWA Animal Biology and CSIRO Livestock Industries

in Floreat, thanks for help in the animal house, with lab work, for volunteering for those

terrible trips to Tammin to collect saltbush, and for dragging me (kicking and screaming

of course) off for a beer every Friday arvo. I would like to thank Peter Hutton in

particular, who was first on my list of volunteers for every experiment.

ix

Thanks to Tony York of Anameka Farm in Tammin for providing a seemingly never-

endless paddock of saltbush to pick, for being so interested in my work and just

generally being a top bloke.

I would also like to acknowledge the support of the CRC for Plant-Based Management

of Dryland Salinity (now the Future Farm Industries CRC), for providing funding for

international conference trips, the (invaluable) postgraduate development program and

continuous media exposure.

Funding for this project and associated conference travel was also gratefully received

from the School of Animal Biology at UWA, CSIRO Livestock Industries, the

Department of Agriculture, Forestry and Fisheries and the Mike Carroll Travelling

Fellowship.

Most of all, I would like to thank my family, for believing in me, and Dan, for being

there all the way.

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1

Chapter 1:

General introduction

Dryland salinity is one of the biggest problems facing Australian agriculture. Most of

the crop and pasture species sown in Australia are salt sensitive and their growth, and

therefore yields and farm profits, are decreased at even low levels of soil salinity.

Salinity directly affects 41% of wool growers nationally, with Western Australia being

the state hardest hit (Land, Water & Wool 2003). Up to 78% of wool growers in

Western Australia have reported that salinity is an issue for them and their long-term

profitability.

Despite the potentially devastating affects of dryland salinity, many farmers remain

optimistic. Up to 70% of wool growers have implemented practices to make their

saline land more productive and profitable (Land, Water & Wool 2003). The most

common practice has been the planting of salt-tolerant pasture or fodder species such as

saltbush.

Old man saltbush (Atriplex nummularia) is one of the most popular species for the

revegetation and rehabilitation of saline land as it can tolerate high levels of soil salinity

as well as extended periods of drought. Saltbush provides a source of feed for sheep

during summer and autumn when the only other alternatives in Western Australia are

poor-quality annual grasses, cereal stubbles, or expensive feed supplements such as

barley grain (Grice and Muir 1988).

The results from lab analyses show saltbush to be a relatively high quality feed. It is

high in nitrogen, moderately digestible and contains relatively low levels of fibre. It

also contains very high levels of sodium and potassium salts (up to 30% dry matter).

However, sheep grazing saltbush tend to lose weight and condition before feed becomes

limiting and this could be caused by their high salt intake.

High intakes of sodium chloride (NaCl) have been reported to have negative effects on

the ruminal environment, microbial populations and rumen fermentation. However,

there is no information available regarding the effects of high levels of mixed salts

(NaCl and potassium chloride (KCl)) on the rumen. Inefficient digestion of diets

2

containing high levels of NaCl and KCl could explain why sheep grazing saltbush

struggle to maintain weight.

It was hypothesised that the poor animal production from saltbush pastures is due to the

negative effects of high NaCl and KCl on the ruminal environment, and subsequent

effects on microbial populations and products of rumen fermentation.

3

Chapter 2:

Review of the literature

2.1 INTRODUCTION

Salt has accumulated in Australian soils over millions of years, but has only become a

problem in agriculture during the last century. Dryland salinity is caused by rising

water tables, which bring dissolved salts to the soil surface. The conventional crop and

pasture species favoured by farmers cannot tolerate the current levels of soil salinity and

farmers have had to turn to new species, such as saltbush, to vegetate parts of their land.

Saltbush species are useful in the revegetation of saline land because they are highly salt

tolerant and produce moderate amounts of reasonable quality feed for livestock

throughout the year. But despite the apparently adequate nutritive value, sheep grazing

saltbush tend to lose weight and condition before feed becomes limiting. This could be

due to the effects of high dietary salt concentrations on the rumen.

In this literature review I will consider the potential of saltbush as forage for sheep and

the possible effects of saltbush on different aspects of rumen function.

2.2 SALTBUSH

Saltbush species (Atriplex sp.) are halophytic shrubs from the family Chenopodiaceae

(Leigh 1986, Grice and Muir 1988). They are found in many arid and semi-arid regions

worldwide, including Australia, North and South America, Africa and Asia. Locally,

Atriplex species are found in the arid and semi-arid zones of central and southern

Australia where they are highly valued by pastoralists for wool production (Leigh 1986,

Grice and Muir 1988, Runciman and Malcolm 1991, Barrett-Lennard and Malcolm

1995, Lefroy 2002).

Saltbushes, particularly old man saltbush (Atriplex nummularia), are also sown in the

Western Australian wheatbelt where they have become an important tool in the

revegetation and rehabilitation of saline land. Old man saltbush is highly tolerant of

4

salinity and drought and can be sown in areas where conventional crop and pasture

species cannot survive (Leigh 1972, Barrett-Lennard and Malcolm 1995, Glenn et al.

1998, Lacey 2001). Not only does old man saltbush survive in these conditions, it

continues to grow and produce green leaves, which can be used as feed for sheep. This

makes saltbush a valuable feed reserve during summer and autumn when the only

alternatives are poor-quality cereal stubbles and annual grasses or expensive feed

supplements like barley grain (Grice and Muir 1988, Runciman and Malcolm 1991).

Saltbush is becoming increasingly popular in Western Australia where farmers are

experiencing a drought, and in many cases, have no other feed available for their stock.

In a review by le Houérou (1992), saltbush stands in the Mediterranean Basin were

reported to produce between five and 20 tonnes of dry matter (DM) per hectare.

However, these results are for total DM production, and include woody stems and

branches as well as the edible leaf material. The edible fraction of the plant is much

lower, and is most likely to be between 0.5 and 1.2 tonnes DM ha-1 (Rashid et al. 1993,

Warren et al. 1994, Morcombe et al. 1996). This may not seem like a lot of feed for

livestock, but there are very few other plants that will grow where saltbush is sown

(Figure 2.1). Saltbush tends to be crash-grazed with large numbers of sheep during

summer and autumn and can support sheep for 500 – 600 grazing days ha-1 saltbush

(Norman et al. 2008).

5

Figure 2.1 Saltbush stand in the Western Australian wheatbelt, April 2006

There is also potential for saltbush to be cultivated in desert environments when

irrigated with saline water (Glenn et al. 1998). Glenn and Watson (1993) estimated that

1.3 million km2 of land worldwide would be suitable for growing halophyte crops under

saline irrigation, including approximately 180 000 km2 in Australia. This could include

coastal deserts irrigated with seawater (e.g. Great Sandy Desert), inland deserts irrigated

with saline ground or surface water (e.g. Lake Eyre Basin) and arid areas irrigated with

saline drainage water (e.g. Western Australian wheatbelt). While most saline

agriculture is currently located on the coastal deserts of Egypt and Saudi Arabia, it may

soon play a role in Australia. Even in the middle of a drought there is plenty of water

available in the shallow (salty) watertables and the ocean (National Land and Water

Resources Audit 2001). This water is too salty for humans or livestock to drink, or to

irrigate conventional crops, but would be invaluable in the cultivation of halophytic

species such as saltbush.

Saltbush plants can tolerate irrigation with water as salty as seawater (~3.5% NaCl).

Ashby and Beadle (1957) grew saltbush seedlings in pots irrigated with 0.3, 1.0, 2.5 and

3.5% NaCl. While the best growth was achieved at 0.3% NaCl, plants survived at all

levels of saline irrigation. Mature saltbush plants are more salt-tolerant than seedlings

Old man saltbush

Volunteer understorey

Bare ground

6

so farmers may be able to increase the level of saline irrigation as their saltbush plots

become more established. In a field situation, Glenn et al (1998) grew almost 50 tons

DM ha-1 when saltbush plants were irrigated with a similar level of saline water (0.4%

total dissolved salts). This production is significantly higher than the value reported

above for saltbush production from saline land and probably reflects the high density of

plants (0.6 m between plants) in the experiment.

2.3 ANIMAL PRODUCTION FROM SALTBUSH

2.3.1 Nutritive value

Compared to the dried annual herbage available in summer and autumn, old man

saltbush is a relatively high quality feed (Table 2.1). It is high in nitrogen, moderately

digestible, and contains relatively low levels of fibre. Importantly, these characteristics

are maintained throughout the year.

7

Table 2.1 Range of nutritive values of Atriplex nummularia in the literature compared

to a high quality pasture (lucerne) or poor quality roughage (oaten-hay) (Beadle et al.

1957, Wilson 1966a, 1966b, Weston et al. 1970, Leigh 1972, Hassan et al. 1979,

Hassan and Abdel-Aziz 1979, Davis 1981, Ostrowski-Meissner 1987, Watson et al.

1987, Arieli et al. 1989, Kessler 1990, Warren et al. 1990, El-Hyatemy et al. 1993,

Abou El Nasr et al. 1996, Chriyaa et al. 1997a, Chriyaa et al. 1997b, Ben Salem et al.

2002a, 2002b, Norman et al. 2004, van der Baan et al. 2004).

Feed component (% DM) Saltbush Lucerne Hay

Digestibility (in vitro) 59 – 82 68 64

Digestibility (in vivo) 34 – 74

Crude protein 9 – 22 16 4

Neutral detergent fibre 34 – 60 34 59

Acid detergent fibre 14 – 38 44 31

Ash 15 – 35 9 3

Na 4 – 8 < 1 < 1

K 1 – 4 3 < 1

Mg 1 – 5 < 1

Ca 1 – 7

Cl 6 – 14

Oxalates 2 – 9

Saponins 5

Nitrate < 0.1

Tannins < 0.1

Saltbush also contains high levels of ash (Table 2.1), which has no energy value, but

can contribute to the apparent digestibility of the feed (Chriyaa et al. 1997a, Masters et

al. 2001). Digestibility (dry matter digestibility or organic matter digestibility) is the

proportion of feed eaten that is not excreted in the faeces. It is calculated by measuring

feed intake and faecal output. In most cases the measured or ‘apparent’ digestibility

slightly underestimates the ‘true’ digestibility of a diet because faecal output contains

endogenous contributions (e.g. sloughed off intestinal cells and microbial matter) that

8

have not been accounted for. With saltbush, soluble components of the ash are

absorbed from the rumen, increasing the apparent digestibility of the diet.

High levels of ash may also account for the differences observed between the in vivo

and in vitro digestibility of saltbush. While in vitro techniques such as pepsin-cellulase

digestions are cheaper and less labour intensive than measuring in vivo digestibility,

they produce results of varying accuracy (de Boever et al. 1988, van der Baan et al.

2004). In the case of saltbush this is probably because the in vitro techniques fail to

account for changes in the rumen when sheep are fed high-salt diets – most notably, a

reduced residence time for feed particles in the rumen, which would increase the

proportion of undigested feed excreted in the faeces (Hemsley et al. 1975).

Most of the ash in saltbush is salt, in particular NaCl and KCl (Table 2.1). Salt levels

are highest in the saltbush leaves during summer. For example, Wilson (1966b) found

that A. nummularia contained only 14.7% Na, K and Cl (DM basis) in winter, compared

to 21.6% in summer. Because sheep usually graze saltbush in summer and autumn, this

means that they will consume high levels of NaCl and KCl.

The daily requirements of sheep for salt are 0.7 – 0.9 g Na, 5 - 8 g K, and 0.25 – 1.0 g

Cl kg DM-1 (Standing Committee on Agriculture 1990). Sheep grazing saltbush

consume amounts of salt that are well in excess of their daily requirements. The most

common side effects of high salt intakes are a reduction in feed intake and lower weight

gain, though high K intakes can sometimes cause hyperkalaemia (K toxicity) and

cardiac arrest (Underwood and Suttle 1999). High K intake can also cause

hypomagnesaemia (Mg deficiency) in ewes by reducing the absorption of Mg from the

rumen (Standing Committee on Agriculture 1990). However, hypomagnesaemia is

unlikely to occur in sheep grazing saltbush as the Mg content of saltbush is several

times the daily requirement of 1.2 – 1.8 g kg DM-1 (McDowell 1992).

2.3.2 Feed intake

The voluntary feed intake of a forage is influenced by its availability, palatability (taste,

odour and texture) and physiological limitations, for example, the size of the rumen and

rate of organic matter clearance. Many authors have reported that domestic livestock

9

tend to select poor-quality annual herbage or perennial grasses when grazing saltbush

pastures, despite the higher nutritive value and dry matter availability of saltbush (Leigh

1986, Grice and Muir 1988, Runciman and Malcolm 1991, Masters et al. 2001).

Thomas et al (2007a) found that sheep will select both high and low salt feeds to

improve the feeding value of their diet, and this may explain the feeding behaviour of

sheep grazing saltbush. Sheep always consumed a low-salt alternative when it was

offered and they increased their intake of the low-salt feed when it was of higher

quality.

The low voluntary feed intake of sheep grazing saltbush is probably due to the large

amount of salt in the leaves (Table 2.1). When very small amounts of salt (around 1%)

are added to a diet, feed intake increases (McClymont et al. 1957, Campbell and

Roberts 1965, Chiy et al. 1993, Chiy et al. 1994). When more salt is added to either the

diet or drinking water, feed intake decreases (Meyer and Weir 1954, Peirce 1957, 1959,

Weeth and Haverland 1961, Wilson 1966c, Wilson and Hindley 1968, Ternouth and

Beattie 1971, Bergen 1972, Moseley and Jones 1974, Hemsley 1975, Hemsley et al.

1975, Kato et al. 1979, Rogers et al. 1979, Phillip et al. 1981, Rossi et al. 1998, Masters

et al. 2005). This is accompanied by an increase in the time taken to consume the ration

– resulting from a decrease in the size and frequency of meals (Hemsley 1975, Rossi et

al. 1998).

The reduction in feed intake appears to be a response to the increase in rumen

osmolality. Several authors have increased the osmolality of rumen fluid using volatile

fatty acids, polyethylene glycol and silage extracts instead of salt (Ternouth and Beattie

1971, Phillip et al. 1981, Carter and Grovum 1990). In all cases, feed intake was

decreased by the same amount as it was when NaCl or KCl was added to the rumen.

When the osmolality of the abomasum was increased there was no effect on feed intake

(Carter and Grovum 1990).

The mechanism by which an increase in rumen osmolality causes a decrease in feed

intake is still largely unknown. Bergen (1972), Martin and Baile (1972), and Carter and

Grovum (1990) attribute the decrease in feed intake to neuronal osmoreceptors in the

rumen wall, although no one has been able to identify any (Forbes and Barrio 1992).

Bergen (1972) and Martin and Baile (1972) were able to reverse the effects of rumen

osmolality on feed intake by infusing 20 mg of local anaesthetic (Carbocaine) into the

10

rumen to suppress the activity of the osmoreceptors. The infusion of other local

anaesthetics (Xylocaine/Lidocaine or Oxethazaine) evoked no response in feed intake

(Martin and Baile 1972, Carter and Grovum 1990). This is most likely due to the more

rapid onset and prolonged action of Carbocaine.

Rumen osmolality could also control voluntary feed intake through the endocrine

system. Blache et al (2007) measured the effects of a high NaCl (20% DM) diet on the

endocrine control systems that regulate feed intake – namely plasma concentrations of

leptin, insulin and cortisol. Contrary to expectations they measured no change in leptin

and cortisol concentrations in response to salt intake, and a decrease in insulin

concentration. This would usually cause an increase in feed intake, so it was concluded

that these hormones do not play a major role in the control of feed intake in sheep

consuming high salt diets. An alternative hormone for the control of feed intake in

sheep fed salty diets could be vasopressin. This hormone is released in response to

plasma hypertonicity and has been shown to reduce feed intake in goats (Meyer et al.

1989).

Alternatively, the aversion of sheep to saltbush could be due to the high levels of

secondary compounds found in the leaves (Table 2.1). Saltbush contains oxalates,

saponins, nitrates and tannins, which have been shown to decrease the voluntary feed

intake of sheep fed other diets (Harborne 1991, Burritt and Provenza 2000, Mueller-

Harvey 2006). This could be through reduced palatability or post-ingestive feedback.

The rate of clearance of organic matter from the rumen is unlikely to affect the intake of

saltbush. When feed accumulates in the rumen distension causes satiety signals to be

sent to the central nervous system, and feed intake is reduced (Weston 1996). The

moderate digestibility (Table 2.1) and high water intake of sheep fed saltbush means

that most saltbush would pass through the rumen faster than other diets. This means

that organic matter would not get a chance to accumulate in the rumen so there would

be plenty of room for more feed and intake would not be reduced. Hemsley et al (1975)

reported that water does not accumulate in the rumen so is unlikely to affect rumen

distension and voluntary feed intake.

11

2.3.3 Water Intake

The effects of salt intake on water intake are well documented, and even small amounts

(as little as 1%) of salt in the diet of sheep and cattle causes an increase in water

consumption (Meyer and Weir 1954, Peirce 1957, 1959, Weeth et al. 1960, Peirce

1963, Wilson 1966c, Wilson and Hindley 1968, Potter et al. 1972, Moseley and Jones

1974, Hemsley 1975, Tomas and Potter 1975, Cheng et al. 1979, Rogers et al. 1979,

Carter and Grovum 1990, Rossi et al. 1998, Masters et al. 2005). As a general rule, the

more salt that is added to the diet the more water is consumed. Sheep grazing saltbush

will drink up to eight litres of water per kg DM intake (Wilson 1966b, Arieli et al. 1989,

Atiq-Ur-Rehman et al. 1994, Casson et al. 1996). It is physically impossible for a

sheep to drink this much water at once and sheep grazing saltbush drink more

frequently than those fed stubbles in order to consume the extra water required.

Because of this farmers may need to install extra watering points in their saltbush

pastures so that sheep waste less time and energy walking to and from watering points

rather than grazing.

When salt is added to the drinking water the same increases in water consumption do

not occur. Salty drinking water is reasonably common in Australian underground water

supplies and in surface water from areas affected by salinity. When drinking water

contains moderate salt concentrations (<1.3%) the amount of water consumed by

livestock increases relative to the amount of salt in the water (Weeth et al. 1960, Weeth

and Haverland 1961, Wilson 1975). However, when the salt concentration of water is

above 1.3%, water intake is reduced. This in turn can limit the amount of feed the

animal consumes, especially if the feed also contains high amounts of salt, like saltbush.

The most comprehensive series of experiments where the tolerance of sheep to saline

drinking water was examined were by Peirce (1957, 1959, 1960, 1962, 1963). He

investigated salts commonly found in underground water supplies used by livestock –

NaCl, magnesium chloride (MgCl2), calcium chloride (CaCl2), sodium sulphate

(Na2SO4), sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3). The salts

were combined in various combinations, with the total salt concentration of water being

around 1.3%. In all cases, adding salt to the drinking water increased the amount of

water consumed by the sheep. This was exacerbated by high temperatures (>25˚C).

We would expect sheep to experience these high temperatures during the times of the

12

year when they are grazing saltbush. The addition of MgCl2 or CaCl2 to water already

containing NaCl caused a further increase in water intake. This was not seen with

Na2SO4, Na2CO3 or NaHCO3.

2.3.4 Liveweight gain

Saltbush shrublands are considered to be among the most productive natural pastures in

the Australian rangelands. However, in most experiments involving sheep grazing pure

saltbush stands the sheep lose weight and condition before feed becomes limiting

(Casson et al. 1996, Morcombe et al. 1996, Hopkins and Nicholson 1999). This could

be because sheep prefer to eat the poor quality annual herbage in preference to saltbush,

or due to the extra energy requirements associated with walking to water, grazing, and

coping with environmental stresses (e.g. Australian summer). However, even in pen-

feeding experiments where sheep do not have a choice of feed, water is readily available

and the environment is controlled, they still often lose weight.

In separate pen-feeding experiments, Abou El Nasr et al (1996) and Atiq-Ur-Rehman et

al (1994) reported that sheep lost weight when fed dried saltbush (A. nummularia and A.

amnicola respectively) ad libitum. While Atiq-Ur-Rehman et al (1994) did not specify

how much weight their animals lost, the sheep in Abou El Nasr’s experiment lost an

average of 109 g day-1. This weight loss was attributed to a salt intake of the animals in

excess of 200 g day-1.

In contrast to this, Wilson (1966a, 1966b) reported that sheep gained 400 g week-1 in a

pen-feeding experiment when they were offered fresh A. nummularia. However, it is

quite likely that these liveweight gains are over-estimates of the potential of saltbush

pastures. The saltbush used in these experiments contained relatively low

concentrations of salt in the leaves (15% DM compared to 27% DM in Abou El Nasr’s

experiment (Abou El Nasr et al. 1996)), and the average daily salt intake was around 50

g day-1. This is significantly lower than the amount of salt consumed by sheep in other

feeding experiments (Atiq-Ur-Rehman et al. 1994, Abou El Nasr et al. 1996). When

the sheep were fed additional salt through the provision of saline drinking water they

lost up to 90 g week-1, suggesting that high salt intakes are the reason for poor animal

performance (Wilson 1966b).

13

2.3.5 Energy balance

The weight losses seen in sheep grazing saltbush could occur at several stages during

the conversion of gross energy to net energy (Figure 2.2). Gross energy represents the

energy potentially available to the animal and is reduced by losses in faecal energy (20-

80% of gross energy). Digestible energy is the energy available from the digestible

fraction of the diet and is reduced by the production of methane and urine (around 19%

of digestible energy). Metabolisable energy is the energy produced during digestion

that the animal is able to use. A small amount of metabolisable energy is lost in heat

production, but the rest (net energy) is available for maintenance, growth and

reproduction.

Figure 2.2 Partition of feed energy (Standing Committee on Agriculture 1990)

Saltbush leaves are reasonably digestible and contain low levels of fibre (Table 2.1), so

we would expect minimal losses of energy during the conversion of gross energy to

digestible energy. However, high levels of ash can reduce digestibility by increasing

the rate of passage of feed through the rumen and may therefore increase losses in

Gross energy

Digestible energy

Metabolisable energy

Net energy

Faecal energy

Methane energy Urinary energy

Heat production

14

faecal energy (Hemsley et al. 1975). Thomas et al (2007b) fed sheep 16 diets

containing four levels of added NaCl (0, 7, 14, 21% DM) and four levels of formulated

organic matter digestibility (proportion of organic matter digested; 55, 62, 69, 76%) in a

4 x 4 factorial design. They found that diets containing 14 or 21% NaCl had

significantly lower in vivo organic matter digestibility than diets containing no salt. The

decrease in digestibility was similar across all four levels of formulated digestibility,

and between ad libitum and maintenance feeding. The authors attributed this decrease

in digestibility to the high water intake of sheep fed salty diets, which increases the rate

of passage of feed particles through the rumen (Hemsley et al. 1975).

Arieli et al (1989) measured the amount of faecal energy lost by sheep fed a

maintenance ration of saltbush (A. barclayana) or a salty diet, both containing around

20% salt, and a control diet containing no salt (Table 2.2). No values for organic matter

digestibility were reported, however, the in vitro dry matter digestibility was the same

(76%) for all diets. Despite this, the authors found that faecal energy losses were

significantly higher in sheep fed saltbush (38% of gross energy) compared to either the

salty or control diets (31% and 29% of gross energy). This is probably due to

differences in the retention time of feed particles in the rumen, which were calculated to

be 9.2, 12.4 and 16.7 hours for the saltbush, salt and control diets respectively. Arieli

offers no explanation for the differences in rumen retention time, but they may be due to

differences in water intake between the diets (which were not measured). Peirce (1959,

1962) found that sheep drank more water when it contained several types of salts.

While it has not been tested, sheep may also consume more water when their diet

contains several types of salts. Because saltbush contains K, Mg and Ca salts in

addition to NaCl, sheep eating saltbush may drink more water compared to sheep fed

diets containing only NaCl. This increase in water consumption would decrease the

retention time of feed particles in the rumen, thus increasing the amount of energy lost

as faecal energy.

15

Table 2.2 Effect of saltbush and salt intake on energy balance in sheep (Arieli et al.

1989)

Diet Energy exchange (kJ / kg bodyweight0.75 per day) Saltbush Salt Control

Gross energy 721 684 718

Faecal energy 273a 210b 210b

Digestible energy 448 473 508

Urinary energy 34.4 43.9 33.1

Methane energy (estimated†) 57.3 57.3 60.7

Metabolisable energy 356b 372ab 414a

Heat production 404a 387ab 359b

Net energy -48.1b -16.3b 54.4a

ab means in rows with different superscripts differ (p < 0.05) †

methane energy was calculated using the equation of Blaxter (1962)

Additional energy losses could occur during the conversion of digestible energy to

metabolisable energy if the effects of saltbush on the rumen increase the amount of

energy lost in methane and urinary energy. In the same experiment mentioned above,

Arieli et al (1989) calculated that energy lost in methane and urine production was 20,

21 and 19% of digestible energy for the saltbush, salt and control diets respectively

(Table 2.2). Urinary energy was measured in urine samples collected over two weeks,

while methane energy was calculated using the equation of Blaxter (1962) (pg 198).

This equation relates methane energy to apparent digestibility, with more methane being

produced from diets with a high apparent digestibility. While there was no significant

difference in the amount of energy lost as urine or methane between the three diets, I

suspect that Blaxter’s equation may not account for high levels of salt in the diet, which

may increase methane production (Mayberry 2003). Arieli et al (1989) measured a

significant increase in the proportion of acetate to propionate in the rumen fluid from

sheep fed both saltbush and the salty diet compared to the control diet. This would

usually be associated with an increase rather than a decrease in methane production, and

we would expect to see more energy lost as methane in the saltbush and salt diets (Van

Soest 1987). Blaxter’s equation (Blaxter 1962) may over-simplify the relationship

16

between methane production and feed composition, particularly when other factors such

as salt content and the presence of secondary compounds can also affect methane

production (see section 2.4.4 of this literature review).

Finally, energy could also be lost during heat production during the conversion of

metabolisable energy to net energy. Arieli et al (1989) calculated heat production from

oxygen consumption using the equation of McLean (1972). Heat production was higher

in the saltbush and salt diets compared to the control diet (Table 2.2), and this was

attributed to the extra heat produced during absorption of sodium from the digestive

tract and excretion by the kidneys. The small but not significant increase in heat

production from sheep fed saltbush compared to the salty diet may be because those

sheep had to absorb and excrete multiple salts, not just NaCl.

Overall, the sheep fed both the saltbush and salt diets in Arieli et al’s experiment (Arieli

et al. 1989) had a net energy deficit (Table 2.2). Because there was no significant

difference in the final energy balance between the saltbush and salt diets the authors

concluded that the high salt content of saltbush was responsible for the poor animal

production. However, there were several differences between the saltbush and salt

diets, particularly in the conversion of gross energy to digestible energy. This indicates

that NaCl alone may not be responsible for the energy deficit of sheep grazing saltbush.

2.4 EFFECT OF SALTBUSH AND SALT ON THE RUMEN

Even when sheep consume adequate amounts of saltbush for maintenance they tend to

lose weight and condition before feed becomes limiting (Wilson 1966a, 1966b, 1975,

Hassan and Abdel-Aziz 1979, Casson et al. 1996, Morcombe et al. 1996). While many

people have attributed this poor animal production to the high salt content of saltbush,

Arieli et al (1989) have been the only group to directly compare saltbush with a

formulated, high-salt diet in the same experiment. Although they observed some

differences they concluded that poor animal production was due to the high level of salt

in saltbush and the effect that this has on rumen fermentation.

Most experiments involving saltbush or salt and ruminants have not been designed

specifically to investigate the effects of salt on the rumen. Despite this, many authors

17

have noted some effects of salt on various rumen parameters as an aside to their main

experimental objectives.

The majority of experiments deal with salt (rather than saltbush), which can be

introduced into the rumen in many ways and forms. In most experiments salt is added

to the daily ration, but it has also been included in drinking water or infused directly

into the rumen through a rumen cannula. The most common form of salt used is

sodium chloride (NaCl), but other Na, K, Ca and Mg compounds have also been used.

In the absence of papers reporting the effects of saltbush on the rumen, this review will

concentrate on the effects of NaCl and, where possible, KCl, as these are the two most

abundant salts in saltbush pastures.

2.4.1 Physiology

Changes in rumen physiology can dictate how the rumen functions. When sheep are

fed diets containing high levels of salt, decreases in saliva production and increases in

the rate of passage of feed particles have major implications for the rumen environment.

This in turn affects microbial populations and rumen fermentation.

Saliva

Saliva in ruminants has two main functions; it lubricates food to assist in mastication

and regurgitation, and buffers the rumen fluid to maintain a constant pH (McDougall

1948). Saliva is normally the main source of Na in the rumen, and usually contains

160-175 mmol Na L-1 (McDougall 1948, Bailey 1961, Michell 1986). Potassium

mainly enters the rumen through the diet, and only small amounts (4-6 mmol L-1) are

found in saliva.

When NaCl is added to the rumen the concentration of Na and Cl ions in saliva

increases (Hemsley et al. 1975, Tomas and Potter 1975, Cheng et al. 1979, Chiy and

Phillips 1993, Chiy et al. 1994). To balance this, the concentration of K ions decreases

and osmolality is not affected. The opposite situation occurs when K is added to the

rumen.

18

Adding salt to the rumen also decreases the flow rate of saliva (Tomas and Potter 1975,

Warner and Stacy 1977). This could help to explain why rumen pH often decreases

when salt is added to the diet (Hemsley et al. 1975, Rogers et al. 1979, de Waal et al.

1989).

Digestion and absorption

When feed particles enter the rumen they are colonised by microorganisms and broken

down. The products of this microbial digestion are removed from the rumen either by

absorption across the rumen wall and into the blood, or by passage to the omasum,

abomasum and small intestine.

High levels of salt intake suppress sheep appetite and increase water consumption. This

increases the dilution of feed particles and microorganisms in the rumen, and the rate at

which they are flushed through the digestive tract (Weston et al. 1970, Potter et al.

1972, Hemsley 1975, Hemsley et al. 1975, Harrison et al. 1976, Thomson et al. 1978,

Cheng et al. 1979, Rogers et al. 1979, Godwin and Williams 1986, Wiedmeier et al.

1987, Arieli et al. 1989). This in turn reduces the efficiency of feed utilisation as small

feed particles, microorganisms, protein and volatile fatty acids pass through the rumen

undigested and unabsorbed.

This change in digestion and absorption was clearly illustrated by Hemsley et al (1975),

who fed sheep a salty (13% DM + 1% salt in water) or control (no salt, fresh water)

diet. The sheep fed the salty diet increased their water intake from 5.4 to 7.5 L day-1,

but there was no significant increase in rumen volume. This was achieved by reducing

the residence time of fluid and digesta in the rumen, as illustrated by a 40% reduction in

the residence time of a radioactive marker (Cr-EDTA) from 20 to 12 hours. They also

recorded an increase in the amount of volatile fatty acids and ammonia leaving the

rumen and abomasum unabsorbed.

Although salt reduces the efficiency of digestion, there is one advantage. Hemsley

(1975), reported increased wool growth (g day-1) when sheep were supplemented with

130 g NaCl day-1 in their feed and drinking water. This was attributed to the high rate

of passage, which increased the amount of undegraded protein available for absorption

19

from the small intestine (Hemsley et al. 1975). This result was supported by Thomas et

al (2007b), who found increases of 16, 18 and 27% in wool growth (corrected for

digestible organic matter intake) when sheep were fed diets containing 7, 14 and 21%

NaCl for two months. While this increase in wool growth efficiency was at the expense

of liveweight gain, these results highlight the potential of saltbush pastures to be used in

profitable animal production systems.

Rumen motility

Contractions and relaxation of the rumen wall move and mix the ingesta. Harding and

Leek (Harding and Leek 1972, Leek and Harding 1975) conducted a series of

experiments in the early 1970s to investigate the control of rumen motility. They found

that the application of 5-30% NaCl solution directly to the rumen wall excited the

epithelial receptors. They suggested that these receptors were involved in the reflex of

rumen contractions. An increase in rumen motility would increase the outflow of

digesta from the rumen, stimulating voluntary feed intake (Forbes 1995).

In more recent experiments, Phillip et al (1981) and Carter and Grovum (1990)

investigated the effects of NaCl loading on rumen contractions. They found that

infusing NaCl into the rumen through a cannula had no effect on the frequency or

amplitude of rumen contractions. However, the maximum salt load used by Phillip et al

(1981) was only 4.5% NaCl, which may not be high enough to evoke a response. The

paper by Carter and Grovum (1990) does not detail the amount of NaCl infused into the

rumen when they measured rumen motility.

In experiments where changes in rumen osmolality were measured in response to a salty

diet or drinking water, maximum osmolalities were less than 400 mosmol kg-1 (Hemsley

et al. 1975, Tomas and Potter 1975). When salts were infused into the rumen through a

cannula to raise the osmolality above normal levels, the maximum osmolality reached

was 810 mosmol kg-1 an hour after the infusion (Bergen 1972). High concentrations of

NaCl (>5% or 1600 mosmol kg-1) applied directly to the rumen epithelium may alter the

frequency and/or duration of rumen contractions (Harding and Leek 1972, Leek and

Harding 1975), but the osmolarity of rumen fluid is unlikely to reach these levels even

when sheep are fed very salty diets.

20

2.4.2 Ruminal microbial environment

The rumen is a well-regulated environment and conditions must be maintained within

narrow limits to ensure normal microbial growth and metabolism. Extremes in rumen

pH and salinity will alter the numbers, types, and activity of microorganisms. This will

affect the amount of energy and waste products produced during rumen fermentation.

pH

The pH of rumen fluid is usually maintained between 6 and 7 through a combination of

saliva production and acid absorption (Van Soest 1987, Theodorou and France 1993,

Underwood and Suttle 1999). Many authors have noted the effects of salt on rumen pH,

but there is no consistent pattern.

Phillip et al (1981), Cardon (1953) and Andrae et al (1995) did not record any

significant change in the pH of rumen fluid when they added NaCl to the rumen. de

Waal et al (1989), Hemsley et al (1975) and Rogers et al (1979) all noted a decrease in

rumen pH, whilst Cheng et al (1979), Godwin and Williams (1986), Rogers et al (1979)

and Wiedmeier et al (1987) observed an increase. Nobody has looked at the effects of

NaCl and KCl combined on rumen pH. Weston et al (1970) are the only group to

measure the rumen pH of sheep fed saltbush and they noted an increase in rumen pH.

Based on the results mentioned above, there does not appear to be a relationship

between rumen pH and the amount or type of salt added, type of ruminant (sheep or

cattle), mode of application (diet, water, rumen infusion or in vitro experiment) or ration

(concentrate or roughage). I have attempted to explain below why the changes in rumen

pH may have occurred.

A reduction in rumen pH could be due to the decreased saliva production in sheep fed

salty diets or changes in the microbial populations (Tomas and Potter 1975, Warner and

Stacy 1977). Several acid-producing bacteria such as Streptococcus bovis and

Selenomonas ruminantium can survive in saline environments, and it is possible that

they dominate the ruminal microbe populations of sheep fed salty diets (Latham et al.

1979, Mackie et al. 1984). Although the effect of high salt intake on reducing the

21

volatile fatty acid concentration of rumen fluid is well documented, the decrease in

rumen pH may be due to an increase in other acids such as lactate. There are no

experiments reporting the effects of salty diets or saltbush on the concentration of

lactate in rumen fluid.

The increase in rumen pH observed by several authors could be explained by a

reduction in volatile fatty acid production and/or concentration in the rumen. A

reduction in the production of volatile fatty acids could be caused by changes in the

microbial populations as a result of increased rumen salinity. In addition, the increased

water intake of sheep fed salty diets could reduce the production of volatile fatty acids

in the rumen by limiting digestion of feed particles. The increased water intake would

also dilute the concentration of any volatile fatty acids produced.

I think that the effects of high water intakes on the concentration of volatile fatty acids,

and indeed, any other acids in the rumen is likely to be the major driver of ruminal pH.

For this reason I think it is more likely that ruminal pH will increase when sheep are fed

a high-salt diet.

It is important for us to understand how and why ruminal pH changes when sheep are

fed saltbush as this will affect the composition and activity of ruminal microbial

populations. The variability in results regarding the effects of NaCl on ruminal pH

indicate that the control of pH may be more complicated than simply a decrease in

saliva or volatile fatty acid production in response to NaCl intake.

Salinity

Rumen salinity is usually maintained at around 250 mosmoles. This level is regulated

by water intake, saliva secretion, the movement of water and salts across the rumen

epithelium and passage through to the lower gastrointestinal tract (Engelhardt 1970,

Van Soest 1987).

Even diets containing small amounts (4% DM) of salt can cause an increase in rumen

osmolality (Odell et al. 1952, Weston et al. 1970, Bergen 1972, Potter et al. 1972,

Warner and Stacy 1972, Hemsley et al. 1975, Tomas and Potter 1975, Warner and

Stacy 1977, Cheng et al. 1979, Rogers et al. 1979, Godwin and Williams 1986, de Waal

22

et al. 1989). The osmolality of the plasma and urine also increases, though to a lesser

extent, as salt is absorbed into the bloodstream and excreted by the kidneys.

When NaCl is added to the rumen the concentration of Na and Cl ions in the rumen

fluid increases and K ions decrease to help balance rumen osmolality (Potter et al. 1972,

Tomas and Potter 1975, Warner and Stacy 1977, Cheng et al. 1979). The reverse is true

for KCl, but there is no information on the balance of ions in rumen fluid when multiple

salts are added to the rumen. Excess Na and K ions are either absorbed across the

rumen epithelium or are flushed further down the gastrointestinal tract.

Most Na and K ions are absorbed from the upper small intestine and excreted by the

kidneys, though there is a small amount of absorption from the rumen (McDowell 1992,

Henry 1995, Committee on Minerals and Toxic Substances in Diets and Water for

Animals 2005). Given the high water intakes and reduced digestion and absorption in

sheep fed salty diets we might expect to see less absorption of Na and K from the rumen

and intestines and more ions excreted in the faeces as is seen in digestive disturbances

such as diarrhoea (McDowell 1992).

Although we know that the salinity of rumen fluid increases when salt is added to the

rumen, we do not know the level of salinity that ruminal microorganisms will be

exposed to in sheep fed saltbush. The saltiest diet used in the experiments listed above

only contained 13% NaCl, while saltbush usually contains in excess of 20% salt

(Hemsley et al. 1975). Rumen salinity may continue to increase as more salt is

ingested, or may reach a level of equilibrium where increased water intake and

absorption of minerals are enough to control rumen salinity. In addition to this, we do

not know if all of the salt in saltbush will be released into the rumen. Playne et al

(1978) reported that different minerals are released at different rates from different plant

materials. Due to the rapid passage of feed particles through the rumen of sheep fed

salty diets it is possible that not all of the salt in saltbush will be released into the rumen

(Hemsley et al. 1975).

23

2.4.3 Ruminal microbe populations

On a “normal” diet rumen salinity is usually around 250 mosmoles, which makes all

rumen microorganisms halophilic (salt-loving). All rumen bacteria and some protozoa

require both Na and K for growth, but they vary in their tolerance to unphysiological

salt concentrations in the rumen fluid (Cardon 1953, Hubbert et al. 1958, Bryant et al.

1959, Quinn et al. 1962, Bergen 1972, Caldwell et al. 1973, Caldwell and Arcand 1974,

Caldwell and Hudson 1974, Latham et al. 1979, Mackie et al. 1984, Javor 1989, Müller

et al. 1990).

Not surprisingly, most rumen microorganisms have maximal growth and production

rates at salt concentrations similar to those normally found in the rumen (Caldwell and

Hudson 1974, Mackie et al. 1984). Small increases in rumen salinity (< 350 mosmol

kg-1) tend to have little effect on the total number or activity of rumen bacteria, and in

some cases the number of bacteria actually increases with small additions of salt

(Campbell and Roberts 1965, Bergen 1972, Thomson et al. 1978, Cheng et al. 1979,

Wiedmeier et al. 1987).

At higher salt concentrations (> 400 mosmol kg-1), especially when K is increased, the

activity and growth of protozoa, Bacteroides sp, and some other cellulolytic bacteria is

inhibited (Quinn et al. 1962, Campbell and Roberts 1965, Bergen 1972, Caldwell et al.

1973, Hemsley et al. 1975, Thomson et al. 1978). Contrary to this, Streptococcus

bovis, Butyrivibrio fibrisolvens, Selenomonas ruminantium and Megasphaera esldenii

can all survive in media hyperosmolar to seawater (Thomson et al. 1978, Latham et al.

1979, Mackie et al. 1984).

In an in vitro experiment, I found that increasing the concentration of NaCl in a

carbohydrate growth medium caused a decrease in the diversity of ruminal microbe

populations (Figure 2.3) (Mayberry 2003). This was illustrated by a decrease in the

number of bands produced in the denaturing gradient gel electrophoresis (DGGE)

analysis in response to increased salinity. Some species of bacteria appear to be more

tolerant of saline conditions than others, and the types of bacteria able to tolerate these

adverse conditions will influence the end products of rumen fermentation.

24

Figure 2.3 Scan of a denaturing gradient gel electrophoresis (DGGE) analysis of DNA

extracted from bacteria grown in a carbohydrate growth medium with added salt. Each

column contains DNA fragments from cultures of rumen bacteria grown at different salt

concentrations (Mayberry 2003). S: laboratory standard, 0-7: % NaCl in cultures.

In addition to the increased salinity of rumen fluid in sheep fed salty diets, the changes

in rumen pH may also affect microbial populations. A good example of this is the

growth of cellulolytic bacteria, which have an optimal pH of 6.7 (Van Soest 1987). A

reduction in rumen pH would reduce the extent of cellulose digestion and increase the

loss of energy from the feed via the faeces.

2.4.4 Products of microbial fermentation

Volatile fatty acids, ammonia and methane are the major end products of microbial

fermentation in the rumen. Volatile fatty acids and ammonia can be used by the sheep

as energy, whilst methane is a waste product. Decreases in the amount of volatile fatty

S 0 1 2 4 5 6 7 S

25

acids and ammonia available for use by the sheep, combined with an increase in

methane production may contribute to poor animal production in sheep grazing

saltbush.

Volatile fatty acids

Volatile fatty acids are fermented from carbohydrates and are a major source of

metabolisable energy to sheep (Van Soest 1987). Normal volatile fatty acid

concentrations in the rumen are between 70 and 130 mM and usually in the proportion

of 63% acetate: 21% propionate: 15% butyrate (France and Siddons 1993). However,

the concentrations of each volatile fatty acid can vary and are affected by the

composition of rumen microbial populations, diet, level of intake, and frequency of

feeding. A decrease in the acetate: propionate ratio increases the efficiency of

metabolisable energy use and microbial protein production. Volatile fatty acids leave

the rumen either through absorption across the rumen epithelium or by passage to the

lower gastrointestinal tract. Absorption of volatile fatty acids from the rumen increases

as rumen pH decreases.

The effects of adding NaCl to the rumen of sheep and cattle on volatile fatty acid

production are reasonably consistent and well documented (Potter et al. 1972, Hemsley

et al. 1975, Harrison et al. 1976, Ward et al. 1976, Kellaway et al. 1978, Thomson et al.

1978, Rogers et al. 1979, Godwin and Williams 1986, Wiedmeier et al. 1987, de Waal

et al. 1989). When large amounts of NaCl (> 5%) are added to the rumen, the following

changes occur;

1. total volatile fatty acid concentration decreases,

2. the acetate to propionate ratio increases, and

3. the absorption of volatile fatty acids across the rumen epithelium is reduced.

These effects are not significant at lower salt intakes.

As mentioned previously, high salt intakes are accompanied by an increase in water

intake and flow rate of liquid and small particles through the rumen. Combined, these

effects may dilute the concentrations of volatile fatty acids in the rumen, and increase

the amount of volatile fatty acids leaving the rumen unabsorbed. Hemsley et al (1975)

measured a significant increase in water intake (from 5 to 8 L day-1) when sheep were

fed a diet containing 13% NaCl plus water containing 1% NaCl compared to a control

26

diet (no salt, fresh drinking water). This reduced the residence time of particles in the

rumen from 20 to 12 hours and increased the flow of liquid from the rumen from 6 to 11

L day-1. The amount of volatile fatty acids leaving the rumen unabsorbed increased

24% from 476 to 592 mmoles day-1. This would mean that sheep consuming salty diets

are not receiving all the energy that is potentially available from the feed (France and

Siddons 1993).

It is interesting to note that in the experiments by Hemsley et al (1975) and de Waal et

al (1989), the addition of salt to the rumen caused a reduction in volatile fatty acid

absorption despite a decrease in rumen pH. Low rumen pH is usually associated with

increased absorption of volatile fatty acids, so it appears that feeding salty diets

increases the rate of passage of liquid and feed particles through the rumen enough so

that rumen pH does not affect volatile fatty acid absorption.

Alternatively, the effects of NaCl on volatile fatty acids could be linked back to the

changes in microbial populations. The decrease in total volatile fatty acid concentration

could be explained by a decrease in the numbers or activity of the rumen bacteria

responsible for volatile fatty acid fermentation. Of these bacteria, it seems likely that

the acetate-producing bacteria are more salt tolerant than propionate-producing bacteria

(Geerligs et al. 1989, Heise et al. 1989). This would explain the increase in acetate at

the expense of propionate.

The exception to this pattern is the experiment of Arieli et al (1989). Arieli et al (1989)

compared the effects of saltbush and a salty diet (described previously) on the rumen,

and found that saltbush had no effect on total volatile fatty acid concentration but the

salt pellet caused a decrease compared to the control diet. The authors suggested that

the difference in volatile fatty acid concentration between the salty diet and saltbush

was due to the solubility of salts from the ration. The presence of more soluble salts in

the diet would cause a greater increase in rumen osmolality and potentially a greater

subsequent increase in water intake, which would dilute volatile fatty acids in the

rumen. If the salt in saltbush was less soluble, the sheep would drink less water, and

there would be less effect on volatile fatty acid concentration. However, the authors

also measured the rumen retention time of the diets, which was 9.2, 12.4 and 16.7 hours

for the saltbush, salt and control diets respectively. The reduced rumen retention time is

probably a result of increased water intake, which would also dilute volatile fatty acids

27

in the rumen. This result contradicts the authors’ explanation for volatile fatty acid

concentration and it is possible that there was an error in the measurement or reporting

of the results.

There are no other experiments measuring the effects of feeding saltbush on ruminal

volatile fatty acid concentrations when compared to a control diet. It is important to

clarify how, and if, saltbush affects volatile fatty acids and this is covered in Chapter

Five of this thesis.

Ammonia

Ammonia is produced during the breakdown of dietary protein in the rumen (Annison et

al. 2002, McDonald et al. 2002). It is then utilised by rumen microorganisms to

synthesise microbial protein. If the diet is low in protein, the concentration of rumen

ammonia will be low and the growth of rumen microorganisms will be slow. As a

result of this the hydrolysis of carbohydrates is retarded. When the ammonia

concentration is high, excess ammonia is absorbed into the blood and converted to urea,

which is excreted. The conversion of ammonia to urea requires energy.

When ruminants consume low salt diets (< 6% NaCl) there is no effect on the

concentration of rumen ammonia when compared to ruminants fed a control diet

containing no salt and the same level of crude protein (Godwin and Williams 1986,

Wiedmeier et al. 1987, de Waal et al. 1989). On high-salt diets, or when sheep are fed

saltbush, rumen ammonia is decreased (Hemsley et al. 1975, Harrison et al. 1976,

Godwin and Williams 1986, Arieli et al. 1989). This is probably due to dilution of

ammonia in the rumen fluid and increased rate of passage due to high water intakes.

Hemsley et al (1975) measured a 23% increase in the amount of ammonia leaving the

rumen (from 5.2 to 6.4 g nitrogen day-1) when sheep were fed a diet containing 13%

NaCl plus water containing 1% NaCl compared to a control diet (no salt, fresh water).

This is very similar to the increase in the amount of volatile fatty acids leaving the

rumen unabsorbed (24%), suggesting that the reduced levels of both volatile fatty acids

and ammonia in the rumen are due to the same cause: increased flow rates.

28

Methane

Methane is a normal byproduct of rumen fermentation and usually comprises about

30% of rumen gases (Quinn et al. 1962, Kay and Hobson 1963). It is expelled from the

rumen by belching and is known by most people for its contribution to greenhouse

gases. In Australia, methane is responsible for 21.2% of national greenhouse gas

emissions, and of this, 12.3% can be attributed to livestock (Australian Greenhouse

Office 2004).

Methane cannot be used by rumen microorganisms or ruminants as an energy source, so

it is considered to be a loss of energy to the animal (Figure 2.1). Up to 12% of

digestible energy can be lost as methane, and it is a major inefficiency in ruminant

production (Bryant 1965, Johnson and Johnson 1995, Ulyatt et al. 1997, Ulyatt et al.

2002).

Methane is produced by a group of microorganisms called methanogenic archaea – or

methanogens. Methanogens are found in a large variety of habitats, including the

rumen, waterlogged soils, sewage, hot springs and sediments (Hough and Danson

1989). They encompass halophilic, thermophilic and mesophilic phenotypes.

A number of methanogens are also halophiles (Hough and Danson 1989). In an in vitro

experiment, Patel and Roth (1977), found that rumen methanogens (Methanospirillum

and Methanobacterium sp.) could tolerate 1.5% NaCl – a higher concentration than that

normally found in the rumen. In non-ruminal habitats, Hough and Danson (1989) and

Sørensen et al (2004), have described species of methanogens that will tolerate up to

22% NaCl. In the case of Sørensen et al (2004), methanogens were found in a solar

saltern in Israel and produced more methane in samples containing higher

concentrations of salt. It is not unreasonable to suggest that rumen methanogens would

also be able to survive and produce methane at high concentrations of NaCl.

I reported that the amount of methane produced in vitro in rumen fluid from sheep fed

saltbush (A. nummularia) was approximately five times higher than in rumen fluid from

sheep fed barley straw or a mixed oaten hay and lupin ration (Mayberry 2003). This

was attributed to the high salt content (25% DM) of the saltbush used. In contrast to

this, Arieli et al (1989) used stoichiometry to calculate the amount of methane produced

29

by sheep fed saltbush (A. barclayana) or a hay-barley diet, both containing around 20%

salt. The methane production was the same for both salty diets, and not significantly

different to that from the control (no-salt) diet. The high methane production in the

experiment by Mayberry (2003) may be because it was performed in vitro, and did not

take into account the increased rate of passage associated with salty diets. High flow

rates may wash the methanogens from the rumen before they can produce much

methane, but they are clearly not inhibited by large amounts of salt in the diet.

It is also worth considering at this point that increasing the salinity of rumen fluid to

380 mosmol kg -1 can reduce the number of protozoa in the rumen (Hemsley et al.

1975). Many species of methanogens live in symbiosis with protozoal populations and

the removal of protozoa from the rumen reduces the number of methanogens in the

rumen (Sharp et al. 1998). An increase in methane production by sheep fed saltbush is

therefore more likely to be attributed to an increase in the numbers or activity of free-

living methanogens.

Given the high level of interest in reducing methane emissions from ruminants (from

both an animal production and environmental perspective), I felt that the production of

methane from salty diets deserved special attention, particularly because it has never

been measured in vivo. If more methane is produced by sheep fed saltbush in vivo as

well as in vitro this would help to explain the poor performance of sheep grazing

saltbush (Mayberry 2003). Methane production by sheep fed saltbush was measured in

vivo in Chapter Five of this thesis.

2.5 OPPORTUNITIES FOR NEW TECHNIQUES IN RUMEN

MICROBIOLOGY

Most of the research where the effects of salt on the rumen have been reported was

carried out in the 1960s and 1970s. Since then there have been significant advances in

the techniques used to measure ruminal microbial populations. This allows us to

investigate the effects of salt and saltbush on the rumen in more depth and with greater

accuracy.

30

Until recently, knowledge of ruminal microbial populations was primarily obtained

using classical culture-based techniques including isolation, taxonomic identification,

enumeration and nutritional characterisation (Hungate 1966, Ogimoto and Imai 1981).

While these techniques are still useful they are limited to ruminal bacteria that can be

cultured out of the rumen and may only account for 10-20% of the microbial population

(Makkar and McSweeney 2005). Theoretically, molecular techniques allow us to

extract DNA from all microbial populations contained in a rumen sample, giving us a

better understanding of how different diets affect ruminal microbial populations.

Although DNA was described in the 1950s, the use of molecular techniques only gained

momentum with the development of polymerase chain reactions (PCR) in the 1980s

(Watson and Crick 1953, Gibbs 1990). PCR is a relatively simple and very fast three-

stage process of thermal cycling (denaturation, annealing and extension) that is repeated

to amplify DNA exponentially (Yu and Forster 2005). The end products of PCR can be

used in many analytical molecular techniques such as denaturing gradient gel

electrophoresis (DGGE).

DGGE is a useful tool for comparing ruminal microbial diversity between sheep fed

different diets or exposed to different treatments (Chiy and Phillips 1993,

Kocherginskaya et al. 2001, McEwan et al. 2005). PCR end-products are run through a

gel containing a chemical gradient (Kocherginskaya et al. 2005). Differing sequences

of DNA from different bacteria will denature at different parts of the gel, leaving a

pattern of bands (see Figure 2.3). This pattern can then be used to identify how similar

bacterial populations are and if they differ in diversity (more bands = more diversity).

Real-time PCR is another useful molecular technique that allows the quantification of

the number of microorganisms in a sample (Heid et al. 1996, Denman and McSweeney

2005, Skillman et al. 2006). DNA from a particular microorganism or group is

amplified using PCR. The difference between real-time and conventional PCR is that

with real-time PCR, the amount of DNA in the reaction is measured after each cycle of

denaturation, annealing, and extension. A fluorescent dye that binds to DNA is added

to the PCR reaction mixture. After each cycle of PCR, the fluorescence of the reaction

is measured and, the more DNA, the stronger the fluorescence. The cycle at which the

fluorescence of the reaction is strong enough to be detected above the background level

31

is called the threshold cycle (Ct) and is used to calculate the number of microorganisms

in the original sample.

The use of these molecular techniques will enable us to measure the effects of salt and

saltbush on complete ruminal microbial populations. We can use DGGE to measure the

effects of salty diets and saltbush on general ruminal microbial diversity, and then use

real-time PCR to measure populations of interest, such as the methanogens.

2.6 PLANT SECONDARY COMPOUNDS

In addition to high levels of salt, saltbush also contains many secondary compounds

(Table 2.1). Secondary compounds often occur in plants as a method of chemical

defence against herbivory (Harborne 1991). They may be bitter tasting, poisonous,

offensively odoured or have antinutritional effects. Environmental stresses can increase

the production of these compounds and may account for the high levels found in

saltbush; a plant that is often subject to water and osmotic stress.

As well as decreasing the voluntary feed intake of saltbush, oxalates, saponins and

nitrates may contribute to poor animal production through their effects in the rumen.

Oxalates are degraded by rumen microorganisms to form Ca-oxalate crystals in the

rumen wall, causing rumen stasis (Burritt and Provenza 2000). Nitrates can be

converted to nitrite in the rumen. Nitrite is toxic to rumen microorganisms and can

reduce cellulose digestion (Van Soest 1987). Saponins can also reduce cellulose

digestion and have been associated with digestive disorders such as bloat and diarrhoea

(Harborne 1991, Burritt and Provenza 2000, Wallace 2004).

In contrast to this, saponins and tannins may actually enhance animal production by

decreasing methane production and protecting protein from rumen degradation (Masters

et al. 2001, Waghorn et al. 2002, Hess et al. 2003, Carulla et al. 2005, Hu et al. 2005,

Mueller-Harvey 2006). High levels of saponins (1-4% DM) have been shown to reduce

methane production in vitro though it is not known if this also happens in vivo (Hess et

al. 2003, Hu et al. 2005). The effectiveness of high levels of tannins (>2% DM) in

reducing methane emissions from ruminants in vivo has been well established although

lower levels (<1% DM) as are found in saltbush (Table 2.1) can actually increase

32

methane production (Sliwinski et al. 2002, Waghorn et al. 2002, Carulla et al. 2005,

Mueller-Harvey 2006). However, in saltbush, the high level of saponins and the

increased rate of passage of feed particles through the rumen may counteract any

increase in methane production caused by the low levels of tannins.

While the concentrations of oxalates, saponins, nitrates and tannins in saltbush are

potentially high enough to limit animal production, we do not know if they contribute to

the poor performance of sheep grazing saltbush. Due to the increased water intake of

sheep fed salty diets, the concentration of secondary compounds in the rumen of sheep

fed saltbush may not be high enough to have any adverse effects on rumen function or

microbial populations (Hemsley et al. 1975). The increased rate of passage may also

flush these compounds through the rumen before they can have any impact.

2.7 USE OF SUPPLEMENTS TO IMPROVE ANIMAL PRODUCTION FROM

SALTBUSH

While sheep struggle to maintain weight when grazing saltbush, the provision of a

supplement such as straw can improve both voluntary feed intake and weight gain. In

an animal house experiment, Warren et al (1990) fed sheep saltbush (A. undulata),

straw, or saltbush + straw (50:50) ad libitum for three weeks. The average DM intake

over the three weeks was 615, 850 and 1449 g day-1 for the saltbush, straw and saltbush

+ straw diets respectively. This increase in feed intake when sheep were fed a

combination of saltbush and straw was reflected by liveweight gain, which was -225,

-25 and 70 g day-1 for the three diets.

The intakes of saltbush and straw are limited by different mechanisms, accounting for

the increase in voluntary feed intake when they are fed in combination. The high level

of salt and secondary compounds in the leaves probably limits the intake of saltbush,

while the intake of straw is limited by rumen fill. When saltbush and straw are fed in

combination, the straw dilutes the amount of salt in the saltbush leaves and slows down

the rate of passage of feed through the rumen allowing more of the saltbush to be

digested. The saltbush provides nitrogen to ruminal microbes, which improves

digestion of the straw. This premise is supported by the results of Nawaz and Hanjra

(1993), who measured an increase in weight gain when goats were fed saltbush (A.

33

amnicola) and dried grass compared to saltbush or grass alone even though there was no

increase in feed intake.

Unfortunately, these results do not translate well to field situations. The two

experiments mentioned above were both animal house experiments and the saltbush and

roughage were combined so that the animals could not substitute one feed for the other.

In field situations sheep tend to eat either saltbush or straw and they fail to gain weight

(Franklin-McEvoy et al. 2007). An alternative would be to feed a high energy,

digestible supplement such as barley that would not limit the intake of saltbush through

rumen fill.

Hassan and Abdel-Aziz (1979) fed sheep saltbush (A. nummularia) ad libitum plus 0,

50, 100 or 150 g barley day-1, equivalent to 10, 20 and 30% of maintenance

requirements, for four weeks. Both voluntary feed intake and weight gain improved

with the barley supplements, though the sheep were not able to maintain weight on less

than 150 g barley day-1. In addition to this, the provision of barley supplements

increased the digestion of crude fibre and protein and reduced the water intake of the

sheep (L kg DM intake-1). Barley provides energy to the rumen microbes to multiply

and convert dietary protein to ammonia, stimulating carbohydrate digestion. Lower

water intakes should mean a slower rate of passage of feed particles through the rumen,

increasing the digestion of saltbush.

van der Baan et al (2004) investigated the effect of feeding different levels of barley on

the digestibility of saltbush (A. nummularia). Sheep were fed a saltbush ration

containing 0, 15, 30 or 45% barley. They found that the inclusion of 15% barley in the

diet increased the digestibility of saltbush, but there was no further increase in

digestibility when sheep were fed diets containing 30 or 45% barley (Figure 2.4). If

there is no further improvement in digestion of saltbush when sheep are fed more than

15% barley, this may be a good indication of the amount of barley needed to maintain

sheep on saltbush pastures.

34

Figure 2.4 Effect of different levels of barley supplements on the organic matter

digestibility (OMD) of saltbush (van der Baan et al. 2004)

The main challenge facing saltbush-grazing systems is getting sheep to grow, or at least

maintain weight, for a minimum cost. The improvement in the digestion of saltbush

when sheep are offered a barley supplement may also be accompanied by an

improvement in the efficiency of rumen fermentation. I would expect to see an increase

in the amount of volatile fatty acids produced, a decrease in the acetate to propionate

ratio, and lower methane production. If the improvement in rumen fermentation

followed the same pattern shown in Figure 2.4, we may be able to use this information

to recommend a level of barley supplementation to producers. This practical

application of my research is discussed in Chapter Six of this thesis.

2.8 SUMMARY

There is clearly potential for saltbush to provide animal production from salt-affected

land. Not only is saltbush tolerant of salt and drought, it can also provide a reasonable

quality diet for sheep throughout the year. It is particularly useful during droughts,

0

20

40

60

80

0 10 20 30 40 50

% barley in diet

OMD (%)

35

when alternative feeds are scarce or expensive. The problem facing producers is that

the level of animal production from saltbush is lower than expected given the expected

nutritive value.

It has been assumed that the poor production of sheep grazing saltbush is entirely due to

the high salt content of the feed. However, most experiments have only measured the

effects of high NaCl intakes on animal performance. Saltbush also contains several

other salts, in particular KCl. Arieli et al (1989) have been the only group to compare

directly the effects of saltbush and high-NaCl diets, and they found several differences.

These differences may be negated by the addition of KCl to the diet.

In their experiment, Arieli et al (1989) demonstrated that there were differences

between saltbush and a formulated high-salt (NaCl only) diet in the conversion of gross

energy to net energy. There was a greater loss of energy as faecal energy and heat

production in sheep fed saltbush compared to the sheep fed the salty diet. This meant

that the sheep fed saltbush had a greater net energy deficit compared to sheep fed the

salty or control diet. Sheep fed diets containing NaCl and KCl may need to drink more

water, causing an increase in the rate of passage of feed through the rumen and loss of

energy in the faeces. Mixed salts may also cost more energy to absorb and excrete,

accounting for the differences in heat production between the two diets.

Arieli et al (1989) also reported differences in volatile fatty acid production between

saltbush and high NaCl diets but were unable to provide a plausible explanation.

Saltbush also contains secondary compounds and, while their presence cannot be

overlooked, I think that the effect of mixed salts on the rumen may be more important

in explaining the poor production of sheep grazing saltbush. It is likely that high water

intakes will dilute the concentration of secondary compounds in the rumen, so that they

will not be present in high enough concentrations to have much effect. In addition to

this, the increased rate of passage of feed particles through the rumen may mean that not

all of the secondary compounds are released into the rumen fluid.

The experiments in this thesis were designed to test the general hypothesis that poor

animal production from saltbush pastures is due to the negative effects of high NaCl and

36

KCl on the ruminal environment (pH and salinity), and subsequent effects on microbial

populations and products of rumen fermentation (volatile fatty acids and methane).

Feeding diets high in salt or saltbush increases the salinity of the rumen fluid and this is

likely to be one of the major factors influencing microbial populations and rumen

fermentation. However, the values given for rumen salinity in the literature are either

from a single rumen sample (e.g. three hours after feeding) or are averages from several

samples. This gives no indication of the extent that the salinity of rumen fluid varies

when sheep are fed salty diets. There is also no information indicating whether all the

salt in saltbush is released into the rumen during digestion. Due to the increased rate of

passage of feed particles from salty diets, some of the salt may be retained in the

saltbush when it moves onto the omasum and abomasum. This is important because the

level, variation and types of salt in the rumen will influence the types of ruminal

microorganisms able to survive.

There is also a lack of consistency in regard to the effects of salty diets or saltbush on

rumen pH. This can be a limiting factor for microbial populations, and influences the

absorption of volatile fatty acids from the rumen. There is only one experiment where

the rumen pH of sheep fed saltbush was recorded and it increased compared to other

diets (Weston et al. 1970). Rumen pH from sheep fed salty diets has been measured

several times but there is no relationship between the amount or type of salt added and

the effect on rumen pH. It is important to clarify this so that we can establish if the high

salt content is responsible for the increase in rumen pH measured in sheep fed saltbush,

or if it is caused by some other factor, such as the presence of secondary compounds.

The changes in the ruminal environment caused by salt and/or saltbush will influence

the composition and activity of microbial populations. There have been no in vivo

studies where changes in microbial populations in response to feeding saltbush have

been measured. It is essential for us to understand how the ruminal microbial

population changes in sheep fed saltbush if we are to manipulate rumen fermentation or

plan appropriate supplementation strategies to improve animal production. Recent

advances in ruminal microbiology make it possible for us to do this.

37

In my first experiment I tested the hypothesis that high levels of salt (NaCl and KCl) in

saltbush alter the ruminal environment by increasing the salinity and pH of rumen fluid,

and that these changes would alter the composition of the ruminal microbial population.

Following on from this, there is a lack of information regarding the effects of feeding

saltbush on the production of energy and waste products in the rumen. It is well known

that high levels of NaCl in the diet reduce volatile fatty acid concentrations in the

rumen, but it is possible that feeding saltbush will have no effect on volatile fatty acid

concentrations. The in vivo production of methane by sheep fed saltbush or high salt

diets also needs to be examined given the high levels of methane produced in vitro in

rumen fluid from sheep fed saltbush and the significance of methane to animal

production and greenhouse gas emissions (Mayberry 2003).

In my second experiment I tested the hypothesis that sheep fed saltbush would have less

efficient rumen fermentation than sheep fed a control diet and that the changes in rumen

fermentation would be due to the high concentration of NaCl and KCl in the diet.

From a more practical perspective, farmers need to know how to manage changes in the

rumen of sheep fed saltbush in order to stop their livestock from losing weight. The

best option currently available is to feed a high-energy supplement such as barley.

Barley improves the performance of sheep grazing saltbush by improving rumen

fermentation. However, barley grain is expensive and farmers need to know how much

barley their livestock require to maintain weight on saltbush pastures. van der Baan et

al (2004) reported that there was no further improvement in the digestibility of saltbush

when more than 15% barley was included in the diet. Changes in rumen fermentation

may follow the same pattern.

In my final experiment I tested the hypothesis that there would be an optimal amount of

barley required to improve the efficiency of rumen fermentation in sheep fed saltbush.

38

Chapter 3:

General materials and methods

3.1 EXPERIMENTAL DESIGN

The main hypothesis tested in this thesis was that poor animal production from saltbush

pastures is due to the negative effects of high NaCl and KCl on the ruminal

environment (pH and salinity), and subsequent effects on microbial populations and

products of rumen fermentation (volatile fatty acids and methane). In order to measure

the effects of salt on the rumen, pelleted diets were formulated containing different

levels of salt (0 – 20% DM). Sheep were fed the pelleted diets or saltbush and I

compared ruminal pH, salinity, and microbial populations (Chapter Four) and the

products of microbial fermentation (volatile fatty acids and methane) (Chapter Five)

between the different diets.

In the third experiment (Chapter Six) I measured the relationship between the amount of

barley fed to sheep eating saltbush and the efficiency of rumen fermentation, with the

aim of establishing an optimal level of supplementation. Barley and straw were

substituted for saltbush at 0 – 100% of the maintenance diet and I measured changes in

the ruminal environment and efficiency of rumen fermentation.

Materials and methods common to two or more of the experiments are detailed below.

3.2 ANIMALS

The sheep used in all experiments were 18-month-old merino wethers with no previous

experience of salty diets. New sheep were used in each experiment and the average

weight of all sheep at the start of each experiment was 42.1 ± 0.3 kg.

Liveweight was measured on a weekly basis throughout the experiments. Any animals

that failed to maintain weight due to poor appetite were removed from the experimental

design.

39

Approval for the use of all animals in all experiments was obtained from the UWA and

CSIRO Animal Ethics Committees.

3.3 DIETS

Saltbush (Atriplex nummularia) was handpicked from Tammin (Figure 3.1),

approximately 180 km east of Perth during summer and autumn. Summer and autumn

are when sheep are most likely to graze saltbush. The site where the saltbush was

growing consisted of a brown, shallow, loamy duplex soil, with moderate to high soil

salinity. Salt scalds were evident in some sections of the site. Saltbush leaves and

small stems were slowly air-dried at 50˚C before being fed to the animals.

Figure 3.1 Location of saltbush plot (Tammin, Western Australia)

The non-saltbush diets used in all experiments were manufactured by a commercial feed

processor (Glen Forrest Stockfeeders, Glen Forrest, Western Australia) and pelleted

through an 8 mm die. The control diet (no-salt) used in all experiments consisted of

wheaten chaff, barley, oats, lupins, a mineral and vitamin mix (Siromin; White et al.

1992) and binders (met lime and gypsum) (Table 3.1). The experimental diets used in

the first two experiments were control + 10% salt pellet (low-salt), control + 15% salt

40

pellet (medium-salt – used in first experiment only) and control + 20% salt pellet (high-

salt). The salt used in all pellets was 2NaCl: 1KCl. In the final experiment, I used a

pellet of barley and straw (50:50, dry matter basis).

Table 3.1 Composition of pellets used in experiments. NS: no-salt, LS: low-salt, MS:

medium-salt, HS: high-salt, BS: barley and straw.

Diets

Components (g kg-1 fresh weight) NS LS MS HS BS

Wheaten chaff 525 472 446 420 -

Straw - - - - 488

Barley grain 200 180 170 160 488

Oats 200 180 170 160 -

Lupins 50.0 45.0 43.0 40.0 -

Siromin 10.0 9.00 8.00 8.00 10.0

Binders 15.0 13.5 12.5 12.0 15.0

NaCl - 75.0 113 150 -

KCl - 25.0 37.5 50.0 -

In all experiments, sheep were slowly introduced to the experimental diets to avoid

disruption of normal rumen fermentation. Diets were fed at maintenance for a

minimum of two weeks before sampling to ensure that the sheep had adapted to the new

feed.

Sheep were fed at 0800 h every morning and any remaining feed from the previous day

was collected and weighed.

3.3.1 Feed analysis

Feed samples were ground to pass through a 1 mm screen using a Tecator Cyclone©

mill.

41

The mineral analysis of diets was conducted by a commercial laboratory (CSBP Soil

and Plant Laboratory, Bibra Lake, Western Australia). Dietary cation-anion difference

(DCAD) was calculated using the equation (Na + K) – (Cl + 0.6 S) (Goff et al. 2004,

Charbonneau et al. 2006).

Samples were analysed for total ash by heating to 620˚C for five hours. Neutral

detergent fibre and acid detergent fibre (Goering and Van Soest 1970) were determined

sequentially using an Ankom 200/220 Fibre Analyser (Ankom® Tech. Co.) in

accordance with the operating instructions for this equipment.

3.3.2 Digestibility

In vivo organic matter digestibility of the diets was measured in the second and third

experiments. Sheep were fitted with faecal harnesses for ten days. This included three

or four days of adaptation to the harnesses and six or seven days of faecal collections.

Approximately 10% of the daily faecal output was retained, weighed and oven-dried at

60˚C to determine dry faecal output. The dried faecal samples from each animal were

pooled and approximately 5 g was ground and ashed to calculate organic matter

digestibility: (organic matter intake – faecal organic matter)/organic matter intake.

3.4 RUMEN SAMPLES

The sheep used in the first experiment had a rumen cannula and rumen fluid was

collected through the cannula using a solid plastic pipe.

In the second and third experiment, sheep without a rumen cannula were used. Rumen

fluid was collected using a vacuum pump attached to a stomach tube. The end of the

tube inserted into the sheep had a 1 mm filter so that large feed particles were excluded.

42

3.4.1 Rumen pH and salinity

In all experiments the pH and salinity of rumen fluid were measured immediately

following sampling. The pH of rumen fluid was measured using an Oakton®

Waterproof pH Testr™. Electrical conductivity was used as a measure of ruminal

salinity, and was measured using an Activon Cyberscan CON20. The samples were

then stored and analysed as outlined below.

3.4.2 Volatile fatty acids

Approximately 1 mL of rumen fluid was aliquotted into an eppendorf tube and the pH

adjusted to <3 using 50 µL of concentrated sulphuric acid. Samples were then stored at

-20˚C and sent to a commercial laboratory (Animal Health Laboratory, Department of

Agriculture, South Perth, Western Australia) where they were analysed using high

performance liquid chromatography.

3.4.3 Molecular analysis

Approximately 5 mL of rumen fluid were stored at -20˚C. For extraction of DNA, 1.5

mL of rumen fluid were defrosted and centrifuged at 11 000 rpm for 20 minutes. The

resulting pellet was then washed twice with sterile phosphate buffered saline and

centrifuged at 6000 rpm for 10 minutes. The pellet was then re-suspended in 180 µL of

enzymatic lysis buffer (20 mM Tris-Cl, pH 8.0, 2 mM EDTA, 1.2% Triton X-100) plus

20 mg lysosyme, and incubated at 37˚C for 45 minutes. DNA was then extracted using

the AccuPrep® Stool DNA Extraction Kit (Bioneer, USA).

The use of DNA extractions for denaturing gradient gel electrophoresis and real-time

PCR is outlined in the relevant chapters.

43

Chapter 4:

Saltbush increases the pH and salinity of the rumen microbial

environment

4.1 INTRODUCTION

There is a lack of consistent information regarding the effect of high salt diets on the

rumen microbial environment and microbial populations. Rumen pH has been shown to

both increase and decrease when sheep are fed high salt diets (Weston et al. 1970,

Godwin and Williams 1986, de Waal et al. 1989). A decrease in ruminal pH could be

due to a decrease in saliva production and an increase in the abundance of acid-

producing bacteria in response to high salt intakes (Tomas and Potter 1975, Warner and

Stacy 1977, Latham et al. 1979, Mackie et al. 1984). However, I would expect the pH

of rumen fluid to increase when sheep consume excess salt because the increased flow

rate of liquid and feed particles through the gastrointestinal tract would prevent the

accumulation of acids in the rumen (Hemsley et al. 1975).

Despite this increase in flow rate, feeding diets high in NaCl causes an increase in

rumen salinity. However, there is no information on how diets containing multiple salts

(such as saltbush) affect total rumen salinity and the concentrations of individual ions in

the rumen. It is not known to what extent the rumen salinity and pH varies throughout

the day, or if all the salt in saltbush foliage is released into the rumen.

The aim of this experiment was to clarify the effects of saltbush on the ruminal

environment. Changes in the pH and salinity of rumen fluid will potentially affect the

composition and activity of microbial populations. I hypothesised that the high levels

of salt (NaCl and KCl) in saltbush alters the ruminal environment by increasing the pH

and salinity of rumen fluid, and that this would cause a change in the composition of the

microbial population.

44

4.2 MATERIALS AND METHODS

4.2.1 Experimental design

Twenty-four sheep (average weight 44.7 ± 0.6 kg) were penned individually in the

animal house for 11 weeks. This consisted of a two-week quarantine period, four weeks

for fistulation surgery and recovery, and a five-week experimental period.

During the experimental period sheep were fed diets containing salt (NaCl and KCl) or

saltbush. Rumen samples were taken over a 24-hour period at the end of the experiment

to measure the effects of salt and saltbush on rumen pH, salinity and microbial

populations.

An in vitro experiment was also performed to establish if the changes in rumen pH were

a response to the increase in rumen salinity or changes in microbial fermentation.

4.2.2 Establishment of a rumen cannula

The fistulation surgery was performed in two stages, approximately 10 days apart

(Hecker 1974). The first stage involved creating a hernia by suturing the rumen wall to

the abdominal wall and was performed under general anaesthetic. The second stage

was performed under a local anaesthetic and involved the installation of a rumen

cannula.

The sheep were allowed to recover for two weeks following the surgery before being

introduced to the experimental diets.

4.2.3 Diets

The diets offered to the sheep were the control pellet (no-salt), control + 10% salt pellet

(low-salt), control + 15% salt pellet (medium-salt), control + 20% salt pellet (high-salt),

or air-dried old man saltbush (Atriplex nummularia) containing approximately 20% salt

45

(Table 3.1 and 4.1). The sheep were offered a maintenance ration, but did not consume

it all on the low-salt, medium-salt, high-salt or saltbush diets.

The sheep were randomly allocated to the diets and were gradually introduced to them

over two weeks before being fed 100% of the diets for a further three weeks. Several

sheep refused to eat the salty diets, and were removed from the experiment. This left

four animals in each group.

Table 4.1 Nutritive value of the experimental diets. NS: no-salt, LS: low-salt, MS:

medium-salt, HS: high-salt, SB: saltbush.

Feed component Diet

(% DM) NS LS MS HS SB

Na 0.28 3.30 4.28 5.45 7.27

K 0.86 1.96 2.59 3.33 3.33

Cl 0.73 6.38 7.88 10.2 10.3

Ash 5.46 14.7 20.8 25.0 31.2

N 1.62 1.38 1.32 1.22 1.60

Neutral detergent fibre 50.7 45.8 47.6 40.0 30.0

Acid detergent fibre 23.3 21.0 18.5 20.2 15.0

DCAD† (mEq kg DM-1) 44.4 58.0 218 265 902 †

DCAD: dietary cation-anion difference

4.2.4 Rumen fluid collection

On the final day of the experiment, 10 ml of rumen fluid was collected from each of the

sheep via the rumen cannula at 0800 h just prior to feeding. Following feeding,

additional 10 ml rumen fluid samples were collected every hour for the first 12 hours

and then every two hours for the following 12 hours. The pH and electrical

conductivity of each sample were measured immediately and the sample was then

frozen and stored for further analysis.

46

4.2.5 Concentration of Na and K ions in rumen fluid

The concentration of Na and K ions in the rumen fluid were measured using Atomic

Absorption Spectrometry (Perkin Elmer, Aanalyst 300) using the standard operating

procedure for the equipment. Sodium was measured in emissions mode (λ 589) and K

in absorption mode (λ 766.5).

Mixed element standards of Na and K were used, and all standards and samples of

rumen fluid were prepared in 1000 mg L-1 cesium (as cesium chloride in milli-Q water),

which acts as an ionisation suppressant. The rumen fluid samples were diluted to

1/1000, and where necessary 1/2000, to allow measurement within the linear range of

the instrument and calibration curve.

4.2.6 Analysis of microbial populations

I had originally intended to compare microbial diversity between all animals at all

sampling times, but I had difficulty optimising the DGGE technique. I was unable to

obtain a clear banding pattern on the DGGE gels despite experimenting with several of

the parameters involved in the procedure (e.g. washing of rumen fluid before DNA

extraction, urea concentration gradient in gel). In the end, I was only able to compare

ruminal microbe populations between sheep for the samples taken three hours after

feeding. I used DNA from three sheep from each diet so that all the samples could be

run on the same gel to improve the accuracy of the comparisons. The methods used for

this gel are described below.

DNA was extracted (see section 3.4.3) from the rumen fluid samples taken three hours

after feeding on the final day of the experiment. The variable V3 region of 16S rDNA

was amplified using polymerase chain reactions (PCR) with Muyzer primers and a

HotStarTaq® DNA Polymerase Kit (QIAGEN) (Muyzer et al. 1993). The PCR reaction

mixture contained 1x QIAGEN PCR buffer, 2.0 mM MgCl2, 200 µM of each dNTP, 20

pmol of each primer, 2.5 µL Taq DNA Polymerase and 5 µL of genomic DNA (per 50

mL reaction). The final volume was adjusted with sterile nuclease-free water. The

47

region was amplified using “touch down” PCR, with the DNA polymerase being added

before thermal cycling (Simpson et al. 1999). The cycling consisted of heat-activation

of Taq-polymerase at 95˚C for 15 minutes, followed by 20 cycles of a three-step

process of denaturation at 94˚C for one minute, annealing at 65 - 55˚C (decreased by

1˚C every second cycle) for one minute, and extension at 72˚C for one minute. Once

the annealing temperature had dropped to 55˚C, nine additional cycles of denaturation,

annealing and extension were performed. Final extension was done at 72˚C for 10

minutes.

PCRs were run in an acrylamide gel with a urea concentration gradient of 40 to 60% at

60˚C and 100 V for 17 hours using the Bio-Rad DCode™ Universal Mutation Detection

System. The gel was then stained using a 1/10 000 dilution of SYBR®Gold nucleic acid

gel stain (Invitrogen) for 15 minutes and destained with de-ionised water. Gel images

were obtained using an Infinity 3000 gel documentation system (Vilber Lourmat).

Our laboratory standard was also run on the DGGE gel in three columns to allow me to

account for any ‘smiling’ (drooping edges) in the gel when comparing the position of

bands in different columns. The standard was prepared from a mixture of bacterial

cultures (Butyrivibrio fibrisolvens, Prevotella ruminicola, Fibrobacter succinogenes,

Streptococcus Bovis, Lactobacilus sp. and Klebsiella sp.) grown in carbohydrate media

in the lab. DNA extraction and PCR amplification were performed as for the

experimental samples.

There were not enough bands of high enough intensity in the final gel for me to analyse

the gel using the software available in our lab (Kodak 1D Image Analysis software,

version 3.6, Eastman Kodak Company). This meant that I was unable to compare the

position and intensity of the bands in the gel. Instead, to obtain some information from

these samples I counted the number of bands that I was able to identify and compared

the diets using analysis of variance (see section 4.2.8).

48

4.2.7 In vitro experiment

To establish if the salinity of rumen fluid was the sole driver of rumen pH, I added salt

to clarified rumen fluid from sheep fed a non-salty diet and measured the electrical

conductivity and pH.

Frozen rumen fluid was thawed and centrifuged at 12,000 and 14,000 rpm for 20

minutes each at 5°C. This separated the bacteria and feed particles from the liquid

fraction of the rumen fluid, but should not have affected the buffering capacity. The

supernatant was collected and used for the experiment.

NaCl and KCl were added to the clarified rumen fluid to create a concentration gradient

of 0 to 5000 mg L-1 Na and K, which was the range observed in rumen fluid from the

sheep fed the salty diets. The pH and electrical conductivity were measured once the

salts had dissolved in the rumen fluid.

4.2.8 Statistical analyses

A one-way analysis of variance with Tukey’s pairwise comparisons was used to

determine the effects of diet on pH, electrical conductivity, Na and K concentration, and

microbial populations. Analyses were conducted using the Genstat statistical package

(Genstat 2005).

4.3 RESULTS

4.3.1 Rumen pH

Over the 24-hour sampling period, the average pH of rumen fluid from sheep fed the

low-salt, medium-salt and high-salt diets was lower (p < 0.05) than the average pH of

rumen fluid from sheep fed the no-salt (control) or saltbush diets (Table 4.2). However,

there was no relationship between rumen pH and the amount of salt ingested or the

salinity of the rumen fluid. The average pH of rumen fluid from sheep fed the saltbush

diet was higher than the pH of rumen fluid from sheep fed any other diet (p < 0.05).

49

Table 4.2 Effect of increasing salt intake on average (±SE) rumen pH and salinity. The

measures of rumen salinity used are electrical conductivity (EC) and the concentration

of Na and K in the rumen fluid. NS: no-salt, LS: low-salt, MS: medium-salt, HS: high-

salt, SB: saltbush.

Diet

NS LS MS HS SB Salt intake on day of sampling (g) 14 ± 4a 105 ± 9b 138 ± 5bc 171 ± 19c 148 ± 22bc

pH 6.28 ± 0.1a 5.98 ± 0.1b 5.57 ± 0.1c 5.87 ± 0.1b 6.62 ± 0.0d

EC (mS cm-1) 14.1 ± 0.5a 18.1 ± 0.7b 20.6 ± 1.0b 24.7 ± 0.7c 19.6 ± 0.9b

Na (mg L-1) 2305 ± 67a 2376 ± 59ab 2610 ± 69bc 2724 ± 71c 2736 ± 60c

K (mg L-1) 837 ± 25a 1427 ± 32b 1600 ± 51c 1671 ± 37c 1380 ± 32b

abcd means in rows with different superscripts differ (p < 0.05)

In the in vitro experiment, adding salt to clarified rumen fluid had no effect on pH.

There was little diurnal variation in the pH of rumen fluid from sheep fed saltbush

(Figure 4.1). In comparison to this, the pH of rumen fluid from sheep fed the no-salt,

low-salt, medium-salt and high-salt diets dropped after feeding and then slowly returned

to the pre-feeding level. There was no difference in the pre-feeding pH between the no-

salt, low-salt, high-salt and saltbush diets, but they were all higher (p < 0.05) than the

pre-feeding pH of sheep fed the medium-salt diet.

50

0

2

4

6

8

0 4 8 12 16 20 24

Time from feeding (hours)

pH

NS

LS

MS

HS

SB

Figure 4.1 Average pH of rumen fluid (n=4) over 24 hours following feeding. NS: no-

salt, LS: low-salt, MS: medium-salt, HS: high-salt, SB: saltbush.

4.3.2 Rumen salinity

The average electrical conductivity of rumen fluid increased when sheep consumed

more salt (r2 = 0.86), but there was no difference between the low-salt, medium-salt and

saltbush diets (Table 4.2). The amount of salt consumed was better reflected by the

average concentration of Na and K (combined) in the rumen fluid (r2 = 0.98).

4.3.3 Na concentration

The average concentration of Na in rumen fluid was highest in sheep that consumed

more salt (p < 0.05) (Table 4.2). The concentration of Na was higher (p < 0.05) in

rumen fluid from sheep fed the medium-salt, high-salt and saltbush diets compared to

the no-salt diet, and in the high-salt and saltbush diet compared to the low-salt diet.

There was no difference in the pre-feeding Na concentration between diets (Figure 4.2).

Across all diets the Na concentration fluctuated during the day, before returning to pre-

feeding levels after 24 hours.

SE

51

Figure 4.2 Average Na concentration (n=4) in rumen fluid over 24 hours following

feeding. NS: no-salt, LS: low-salt, MS: medium-salt, HS: high-salt, SB: saltbush.

4.3.4 K concentration

The concentration of K in the rumen fluid was much lower (p < 0.05) than the

concentration of Na. Potassium was higher (p < 0.05) in sheep fed salt, but there was

no difference between the low-salt and saltbush, and medium-salt and high-salt diets

(Table 4.2).

As with the Na concentration, the concentration of K in the rumen fluid fluctuated

during the day before returning to concentrations close to those measured before

feeding. The concentration of K in rumen fluid from sheep fed the no-salt diet was

lower (p < 0.05) than the salty diets at all times throughout the day. There was no

significant difference in the pre-feeding K concentration between the low-salt, medium-

salt, high-salt and saltbush diets (Figure 4.3).

0

1000

2000

3000

4000

0 4 8 12 16 20 24 Time from feeding (hours)

Na concentration mg L-1

NS LS MS HS SB

SE

52

Figure 4.3 Average K concentration (n=4) in rumen fluid over 24 hours following

feeding. NS: no-salt, LS: low-salt, MS: medium-salt, HS: high-salt, SB: saltbush.

4.3.5 Microbial populations

The number of bands in the gel tended to increase relative to the amount of salt in the

diet. The rumen fluid from the sheep fed saltbush had the highest number of bands, and

this was significant when compared to the no-salt and low-salt diets (p < 0.05) (Figure

4.4). There was no difference in the number of bands between the no-salt, low-salt,

medium-salt and high-salt diets, and the medium-salt, high-salt and saltbush diets.

0

500

1000

1500

2000

2500

0 4 8 12 16 20 24 Time from feeding (hours)

K concentration (mg L-1)

NS LS MS HS SB

SE

53

Figure 4.4 Image of a DGGE gel of rumen fluid samples taken from sheep fed saltbush

or pellets containing different levels of salt. Each column contains fragments of

bacterial DNA amplified from rumen fluid taken from sheep fed each diet. S:

laboratory standard, NS: no-salt, LS: low-salt, MS: medium-salt, HS: high-salt, SB:

saltbush.

4.4 DISCUSSION

It was hypothesised that the salt (NaCl and KCl) in saltbush would alter the ruminal

environment by increasing the pH and salinity of rumen fluid and that this would

change the composition of the ruminal microbial population. When sheep were fed

diets containing salt or saltbush the salinity of rumen fluid increased relative to the

amount of salt ingested (Table 4.2). Analysis of the microbial populations was limited,

but suggests that adding salt to the diets and feeding saltbush increases ruminal

microbial diversity (Figure 4.4). However, there were differences in rumen pH and

possibly microbial diversity between the salty diets and saltbush, so the hypothesis is

S NS LS S MS HS SB S

54

only partly accepted. This indicates there is something other than salt in saltbush that

affects the ruminal environment and microbial populations.

The results from the in vitro experiment showed that the chemistry of adding salt (NaCl

and KCl) to rumen fluid does not affect rumen pH. However, the reduction in rumen

pH when sheep were fed the salty pellets compared to the control diet (no-salt) indicates

that salt is indirectly responsible for lowering rumen pH (Table 4.2). This could be

caused by a decrease in the amount of saliva produced and consequent reduction in the

rumen buffering capacity of sheep fed salty diets (Tomas and Potter 1975, Warner and

Stacy 1977). Additionally, the changes in rumen pH could be associated with changes

in the microbial populations as a result of increased rumen salinity. Several acid-

producing bacteria (Streptococcus bovis, Selenomonas ruminantium) can survive in

saline environments, and it is possible that they dominate the ruminal microbe

populations of sheep fed salty diets (Latham et al. 1979, Mackie et al. 1984). The effect

of salt on reducing the volatile fatty acid concentration of rumen fluid has been well

documented, but the decrease in pH may be due to an increase in other acids, such as

lactate.

This decrease in rumen pH has several implications for rumen fermentation and animal

production from salty diets. On the positive side, low rumen pH inhibits methane

producers and increases the absorption of volatile fatty acids across the rumen wall

(Schwartz and Gilchrist 1975, France and Siddons 1993). However, these benefits are

far outweighed by the negative effects. A low rumen pH is toxic to many

microorganisms, including cellulose digesters and protozoa, and this is exacerbated by

an increase in rumen salinity (Slyter 1976). As well as the decreased fibre digestion

caused by the reduction in cellulolytic bacteria, low rumen pH can cause a reduction in

volatile fatty acid production, microbial growth, salivation (which would decrease

rumen pH even further) and intestinal motility. The combination of reduced volatile

fatty acid and microbial protein production would be expected to reduce the amount of

energy available to sheep fed salty diets.

The pH of rumen fluid from sheep fed saltbush was higher than the pH of rumen fluid

from sheep fed any of the pelleted diets (Table 4.2), and they may not experience any of

the digestive problems mentioned previously. Because the sheep fed saltbush were

offered a larger volume of feed than the sheep fed the pellets, they probably consumed

55

the saltbush in a series of small meals throughout the day. This constant eating would

be accompanied by constant saliva production, which would help to buffer the rumen

fluid and maintain an elevated pH.

The high pH of rumen fluid from sheep fed saltbush may also be explained by the high

cation-anion balance of the feed (Table 4.1). Diets with a dietary cation-anion

difference (DCAD) value greater than 100 mEq kg DM-1 are alkaline, and associated

with an increase in the pH of blood and urine (Fauchon et al. 1995). They could also

cause an increase in the pH of rumen fluid. While this does not explain the reduction in

rumen pH when sheep were fed salty pellets, it does account for the large difference in

pH between the pellets and saltbush. Alkaline diets are associated with low Ca

absorption, which can lead to milk fever in sheep and cattle (Underwood and Suttle

1999). The high cation-anion balance of the saltbush diet indicates that this diet may

not be suitable for pregnant or lactating ewes as they may develop Ca deficiencies.

My results support the work of de Waal et al (1989) and Hemsley et al (1975) who

noted a decrease in the pH of rumen fluid when sheep were fed diets containing salt,

and Weston et al (1970), who observed an increase in the rumen pH of sheep fed

saltbush. Several other authors (Thomson et al. 1978, Phillip et al. 1981, Godwin and

Williams 1986) either did not record any change in rumen pH, or measured an increase

in rumen pH when sheep were fed diets containing salt. These results could be

explained by the possible contamination of rumen samples with saliva, the use of small

amounts of salt in their experiments, or low feed intake (and therefore low volatile fatty

acid production) by animals fed salty diets.

The pattern of rumen pH in sheep fed the no-salt, low-salt, medium-salt and high-salt

pellets is typical of ruminants fed a single meal (Figure 4.1). The pH drops after

feeding when lactic acid and volatile fatty acids are produced. During the day these

acids are either utilised by microorganisms, or absorbed or flushed from the rumen, and

the pH returns to the pre-feeding value. The lack of diurnal variation in the rumen pH

of sheep fed saltbush probably reflects an eating pattern of several small meals per day

as suggested above.

The low pH of sheep fed the medium-salt pellet compared to the other diets (Table 4.2)

may be explained by the digestibility and fibre content of the ration. For unknown

56

reasons, the medium-salt pellet contained less acid detergent fibre than the other pellets

(Table 4.1). Hence, the medium-salt diet was probably more digestible, which would in

turn lead to higher concentrations of volatile fatty acids in the rumen. After several

weeks of feeding, the pH of sheep fed the medium-salt diet may have stabilised at a

lower base point than the pH of sheep fed the other diets.

The effect of salt intake on rumen salinity was the same regardless of whether the sheep

were fed saltbush or pellets containing salt. In all cases, the salinity of rumen fluid

increased relative to the amount of salt ingested (Table 4.2). Increases in rumen salinity

cause reductions in voluntary feed intake, cellulose digestion and volatile fatty acid and

ammonia production (Ternouth and Beattie 1971, Bergen 1972, Phillip et al. 1981,

Godwin and Williams 1986, Carter and Grovum 1990). Combined, these effects mean

that there would be less energy produced by microbial fermentation in sheep consuming

salty diets.

The increase in rumen salinity could be modelled by the electrical conductivity or

concentration of Na and K in rumen fluid. The electrical conductivity of rumen fluid

reflected the salt intake of sheep fed the salty pellets, but was lower than expected for

the sheep fed saltbush. This is probably because not all the Na and K ions were

released from the plant material (Playne et al. 1978). While the concentration of Na

and K combined better reflected the salt intake of sheep fed saltbush, the electrical

conductivity of rumen fluid may be a more useful measure as it reflects the salinity of

the microbial environment.

Most Na and K ions are absorbed from the small intestines and excreted by the kidneys,

though there can be some absorption from the rumen (McDowell 1992, Henry 1995,

Committee on Minerals and Toxic Substances in Diets and Water for Animals 2005).

Given the high water intakes of sheep fed salty diets, I would expect to see less

absorption of Na and K from the rumen and intestines and more ions excreted in the

faeces as is seen in digestive disturbances such as diarrhoea (McDowell 1992, Masters

et al. 2005).

One of the most interesting results from this experiment was that the concentration of K

in the rumen fluid was higher in sheep fed the salty diets (both pellets and saltbush)

compared to the control diet at all times throughout the day (Figure 4.3). This is

57

unusual because there was no difference in the pre-feeding Na concentration between

any of the diets (Figure 4.2). The sheep is able to remove excess Na ions from the

rumen during the day, but the K ions are allowed to accumulate. Given that most

absorption of Na and K takes place further down the gastrointestinal tract, I would

expect Na and K to be removed from the rumen at the same rate. These results suggest

that Na is being absorbed from the rumen.

When Na and K are absorbed from the rumen, it is by different processes. Potassium

absorption is passive, and down a concentration gradient (Scott 1967). In comparison,

absorption of Na is active, and can be against the electrochemical gradient (Dobson

1959). Stacy and Warner (1966) found that the absorption of Na from the rumen was

related to the osmolality of the rumen rather than just Na concentration. In my

experiment it appears that due to the high rumen salinity (from Na, K and other ions),

Na is actively absorbed from the rumen as well as being flushed further down the

digestive tract. However, because K absorption is passive very little is absorbed from

the rumen until concentrations become very high.

It is difficult to make conclusions confidently about the changes in ruminal microbial

populations based on a single DGGE gel and from a single sampling time-point (Figure

4.4). However, there was a trend for the number of bands in the gel to increase as more

salt was included in the diets, indicating an increase in bacterial diversity. The

microorganisms in rumen fluid from sheep fed the salty diets were exposed to a greater

range of rumen salinities than those in rumen fluid from sheep fed the control diet

(Figures 4.2 and 4.3). A broader range of environmental conditions may accommodate

a larger variety of rumen microorganisms. By the same token, the microorganisms in

rumen fluid from sheep fed the control diet experienced a fairly stable environment, and

were exposed to much smaller changes in rumen salinity. Consequently there was no

reason for this population to diversify.

This is different to my previous results, where increasing the amount of NaCl added to

cultures of rumen bacteria decreased bacterial diversity (illustrated using DGGE, see

Figure 2.3) (Mayberry 2003). However, the salt concentration of the cultures was much

higher (up to 7% NaCl) than I measured in the rumen fluid of sheep fed salty diets.

Moderate increases in ruminal salinity appear to encourage the diversification of

ruminal microbial populations but, under extreme conditions, few species are able to

58

survive. In my earlier experiment, bacterial diversity did not appear to decrease until

the salt concentration of the cultures reached 4% NaCl, and may even have increased

with the addition of 1% NaCl (Mayberry 2003).

The saltbush diet contained the highest number of bands in the DGGE gel, signifying a

greater level of bacterial diversity (Figure 4.4). The saltbush was a more complex diet

than the pellets and the microorganisms would have been exposed to a range of

different substrates (e.g. leaf vs stem) and secondary compounds. In addition, rumen

salinity would have varied throughout the day. Because the pH of the rumen fluid from

sheep fed saltbush was higher than that from sheep fed the other diets, it is also likely

that there would be different microbial species in the rumens of sheep fed the different

diets. This would have been confirmed by the position of the bands in the DGGE gel

had I been able to compare this between diets.

It is unfortunate that I was unable to optimise the DGGE technique to compare

microbial diversity between rumen fluid from sheep fed the different diets. The DGGE

technique has previously only been used in our laboratory to compare ruminal bacteria

grown in cultures. Samples taken from the digestive tract of animals contain inhibitory

substances that can interfere with DNA extraction, PCR and DGGE, and it is possible

that the rumen samples were not adequately washed prior to DNA extraction (McOrist

et al. 2002, Yu and Forster 2005). In support of this, the standards on the gel (Figure

4.4) were prepared from ruminal bacteria grown in a carbohydrate medium and

appeared as seven clear and distinct bands. Bacteria could have been cultured from

rumen fluid from the sheep in this experiment but this would only have enabled

comparison of a small proportion (<20%) of the microbial population (Makkar and

McSweeney 2005).

Based on the changes in ruminal parameters (pH, salinity) and limited evidence of

differences in microbial diversity between sheep fed the different diets, we would

expect to see differences in the products of rumen fermentation. This may depress

animal production from sheep fed saltbush or other salty diets. The effects of feeding

saltbush and high-salt pelleted diets on rumen fermentation in sheep is examined in the

next chapter.

59

Chapter 5:

Saltbush reduces the efficiency of rumen fermentation

5.1 INTRODUCTION

In the first experiment it was demonstrated that feeding sheep saltbush or pellets

containing high levels of salt (NaCl and KCl) increases rumen salinity and may alter the

ruminal microbial population. Changes in the microbial population will affect the end

products of rumen fermentation.

In vitro, rumen fluid from sheep fed saltbush produced five times more methane than

rumen fluid from sheep fed barley straw or a mixed ration of oaten hay, lupins and

minerals (Mayberry 2003). The increase in methane production corresponded to an

increase in the salt content of the feed and the salinity of the rumen fluid. Many

methane-producing archaea (methanogens) are halophilic and, in non-rumen habitats,

produce more methane under more saline conditions (Patel and Roth 1977, Hough and

Danson 1989, Sørensen et al. 2004). Rumen methanogens may also produce more

methane under saline conditions, accounting for the increase in methane production

reported previously (Mayberry 2003). This would be accompanied by an increase in the

ratio of acetate to propionate in the rumen fluid.

However, I conducted this experiment in vitro, with rumen fluid collected from the

sheep and incubated in serum bottles. Consequently, the experiment did not account for

the increase in rumen dilution and flow rate, and reduced digestibility associated with

high salt diets. Both of these factors have been shown to decrease methane production

(Okine et al. 1989, Pelchen and Peters 1998). In an in vivo situation, feed particles may

be washed through the rumen before they can be digested, and there may not be an

increase in the amount of methane produced.

Increased rumen dilution and flow rates have also been shown to decrease the

concentration of volatile fatty acids in the rumen. This is a well-documented response

to feeding high-salt diets, but there is no reliable information as to how saltbush affects

ruminal volatile fatty acid concentrations.

60

Overall, an increase in methane production and the ratio of acetate to propionate in the

rumen of sheep fed saltbush, and a decrease in total volatile fatty acid production would

indicate inefficient rumen fermentation. This may help to explain why sheep fed

saltbush struggle to maintain weight.

I tested the hypotheses that:

1. Sheep fed saltbush would have less efficient rumen fermentation than sheep fed

a control diet (higher methane production, lower total volatile fatty acids, higher

acetate: propionate), and

2. The changes in rumen fermentation would be due to the high level of NaCl and

KCl in the diet.

5.2 MATERIALS AND METHODS

5.2.1 Experimental design

Forty sheep with an average weight of 40.1 kg (± 0.3) were individually penned in the

animal house for 13 weeks. For the first six weeks of the experiment all animals were

fed the control pellet (no-salt) (Table 3.1 and 5.1) at maintenance to enable us to

compare natural variation in methane production between animals. Daily methane

production was measured over a single 23-hour period for each animal during weeks

five and six. Animals were then allocated to one of four treatment diets (no-salt, low-

salt, high-salt, saltbush) (Table 3.1 and 5.1) based on methane production, with average

methane production for each group being between 1.12 and 1.20 L hr-1 kg OM

digested-1. Methane production was measured for a second time after the sheep had

been fed the experimental diets for a minimum of two weeks (weeks 11 and 12).

Numbers of methane-producing archaea (methanogens) and volatile fatty acid

concentration were also measured at this time.

61

5.2.2 Diets

The diets offered to the sheep during the experimental period were the control pellet

(no-salt), control + 10% salt pellet (low-salt), control + 20% salt pellet (high-salt) and

air-dried saltbush containing approximately 16% salt (Table 3.1 & 5.1). Animals were

gradually introduced to the diets during weeks seven and eight, before being fed 100%

of the treatment diets for the remaining five weeks (9-13) of the experiment. They were

offered a maintenance ration, but did not consume it all on the low-salt, high-salt and

saltbush diets. Four sheep were removed from the experiment due to poor appetite,

leaving nine sheep in each group.

Table 5.1 Nutritive value of the experimental diets. NS: no-salt, LS: low-salt, HS:

high-salt, SB: saltbush.

Diets

Feed component (% DM) NS LS HS SB

Na 0.21 3.04 6.11 5.44

K 0.84 2.01 3.50 2.38

Mg 1.36 1.32 1.14 6.65

Ca 7.22 7.82 6.55 4.01

P 2.26 1.97 1.77 1.60

Cl 0.56 5.71 11.3 7.83

Ash 5.40 18.3 27.9 23.6

N 1.54 1.45 1.24 1.99

In vivo organic matter digestibility 66.0 62.8 57.6 47.6

Neutral detergent fibre 53.4 52.1 41.9 36.3

Acid detergent fibre 21.5 20.5 16.9 19.9

DCAD† (mEq kg DM-1) 61.1 138 283 592 †

Dietary cation-anion difference

62

5.2.3 Methane production

The volume and rate of methane production by sheep was measured using four open

system respiration chambers (Klein and Wright 2006). As there were only four

chambers, measurements of methane production took place over ten days, with one

sheep from each group included in each day’s measurements. The sheep were offered

their normal daily ration and immediately enclosed in the chambers at around 0900 h

every morning. They remained in the chambers for approximately 23 hours, and were

removed at around 0800 h the next morning. Methane measurements were corrected for

differences in temperature, humidity, air pressure, feed intake and duration of

measurements between chambers and animals.

During the first four weeks the sheep were trained to the methane chambers for

approximately six hours each day. Sheep were considered “trained” when they ate all

of the feed offered to them in the chambers.

5.2.4 Rumen pH, salinity and volatile fatty acid concentration

When the animals were removed from the methane chambers they were returned to their

pens in the animal house and fed their treatment ration. A sample of rumen fluid was

collected from these animals approximately three hours after feeding using a stomach

tube attached to a vacuum pump. The pH and electrical conductivity of the rumen fluid

were measured immediately following collection, and the sample was then divided up

for measurement of volatile fatty acids and methanogens.

5.2.5 Enumeration of methanogens

DNA was extracted from all rumen fluid samples as described in section 3.4.3. Real-

Time PCR was used to quantify the number of methanogens in the rumen fluid.

Amplification was conducted on an iCycler Thermal Cycler (Bio-Rad, Hercules, CA)

using the method of Christophersen et al (unpublished). Threshold cycles were

calculated automatically by the Bio-Rad iCycler software (version 3.5).

63

5.2.6 Digestibility

Five sheep from each group were fitted with faecal harnesses for the final ten days of

the experiment. Faecal samples were collected during the final six days (week 13) for

measurement of organic matter digestibility (see section 3.3.2).

Ground sub-samples of faeces were sent to the CSBP Soil and Plant Laboratory (Bibra

Lake, Western Australia) for complete mineral analysis. Apparent mineral digestibility

was calculated as (mineral intake – mineral faecal output)/mineral intake, and apparent

absorption as mineral intake – mineral faecal output.

5.2.7 Statistical analysis

A one-way analysis of variance with Tukey’s pairwise comparisons was used to

determine the effects of diet on methane production, populations of methanogens,

rumen pH, rumen salinity and apparent digestion and absorption of minerals. Analyses

were conducted using the Genstat statistical package (Genstat 2005).

5.3 RESULTS

5.3.1 Methane production

Sheep fed saltbush produced 40% more methane per kg of digestible organic matter

intake (DOMI) (p < 0.05) than sheep fed the pellets (Table 5.2). There was no

difference in methane production between sheep fed the no-salt, low-salt and high-salt

pellets.

64

SB

2.04

± 0

.15b

55.3

± 2

0.8b

55.8

± 3

.6ab

77.1

± 0

.58b

15.5

± 0

.60a

15.3

± 1

.09ab

7.47

± 0

.08c

HS

1.41

± 0

.11a

1.7

± 0.

3a

50.6

± 4

.4b

65.2

± 0

.81a

23.5

± 0

.84c

18.3

± 1

.50b

6.48

± 0

.07a

LS

1.56

± 0

.11a

1.9

± 0.

4a

71.3

± 9

.6ab

65.2

± 1

.53a

22.0

± 1

.54bc

15.7

± 2

.58ab

6.03

± 0

.13b

Die

t

NS

1.44

± 0

.13a

3.7

± 2.

6a

84.0

± 1

0.8a

64.1

± 1

.19a

18.9

± 1

.46ab

11.4

± 0

.91a

6.90

± 0

.16a

Tabl

e 5.

2 E

ffec

t of d

iet a

nd ru

men

salin

ity o

n pr

oduc

ts o

f rum

en fe

rmen

tatio

n. N

S: n

o-sa

lt, L

S: lo

w-s

alt,

HS:

hig

h-sa

lt, S

B:

saltb

ush.

M

etha

ne p

rodu

ctio

n (L

hr-1

kg

DO

MI-1

)

Met

hano

gens

(bill

ions

mL

rum

en fl

uid-1

)

Tota

l vol

atile

fatty

aci

d co

ncen

tratio

n (m

mol

L-1

)

Prop

ortio

n of

ace

tate

(%)

Prop

ortio

n of

pro

pion

ate

(%)

Elec

trica

l con

duct

ivity

of r

umen

flui

d (m

S cm

-1)

Rum

en p

H

abcd

valu

es w

ith th

e sa

me

lette

r in

the

sam

e ro

w a

re n

ot si

gnifi

cant

ly d

iffer

ent (

p<0.

05)

65

5.3.2 Methanogens

There were significantly more (p < 0.05) methanogens per mL of rumen fluid from

sheep fed saltbush compared to the control diet (Table 5.2). Adding salt to the pellets

tended to decrease the concentration of methanogens in the rumen fluid, but there was

no significant difference in the numbers of methanogens between the no-salt, low-salt

and high-salt pellets.

5.3.3 Volatile fatty acid concentration

Feeding salt, either as saltbush or pellets, decreased total volatile fatty acid

concentration (Table 5.2), though this was only significant (p < 0.05) in sheep fed the

high-salt pellets.

There was a higher proportion of acetate in rumen fluid from sheep fed saltbush

compared to sheep fed the pellets (p < 0.05) (Table 5.2). There was no difference in the

proportion of acetate in rumen fluid among sheep fed the no-salt, low-salt and high-salt

pellets.

There was a small but not significant decrease in the proportion of propionate in rumen

fluid from sheep fed saltbush (Table 5.2). There was an increase in the proportion of

propionate in rumen fluid from sheep fed the salty pellets, and this was significant for

the high-salt diet (p < 0.05).

The proportion of acetate to propionate in rumen fluid decreased when salt was added to

the pellets (3.4:1, 3.0:1 and 2.8:1 for the no-salt, low-salt and high-salt pellets

respectively) and increased when sheep were fed saltbush (5.0:1).

66

5.3.4 Rumen pH and salinity

Adding salt to the pellets decreased rumen pH (p<0.05), but there was no relationship

between rumen pH and the amount of salt consumed (Table 5.2). The pH of rumen

fluid from sheep fed saltbush was higher than in the rumen of sheep fed the pellets (p <

0.05).

The salinity of rumen fluid was higher in sheep fed salty diets, though this was only

significant (p < 0.05) in sheep fed the high-salt pellet (Table 5.2).

5.3.5 Digestibility

Adding salt to the pellets decreased the digestibility (Table 5.1) of the feed. The

saltbush was less digestible than the pellets.

The amount of Na and Cl apparently digested by sheep fed saltbush was lower than

from sheep fed the salty pellets (p < 0.05) (Table 5.3).

There was a net loss of Mg, Ca and P from the sheep fed saltbush (Table 5.3). There

was no difference in the amount of Mg, Ca and P digested between sheep fed the salty

and control pellets.

67

Table 5.3 Apparent digestion and absorption of minerals by sheep fed diets containing

salt or saltbush. NS: no-salt, LS: low-salt, HS: high-salt, SB: saltbush.

Diets Apparent digestion (%) NS LS HS SB

Na 88.8 ± 1.1a 98.4 ± 0.4b 98.4 ± 0.2b 94.9 ± 0.9c

K 86.6 ± 1.9a 95.2 ± 0.7b 96.7 ± 0.4b 94.7 ± 0.9b

Cl 92.2 ± 0.4a 98.9 ± 0.2b 99.3 ± 0.1b 96.4 ± 0.5c

Mg 29.7 ± 3.6a 10.8 ± 6.1a 10.7 ± 4.3a -19.7 ± 7.0b

Ca -3.02 ± 4.1ab -10.6 ± 10ab 11.4 ± 3.8b -24.0 ± 7.9a

P 13.0 ± 3.9a 29.7 ± 6.3a 43.3 ± 1.2a -46.2 ± 16.4b

Apparent absorption (g/day)

Na 1.89 ± 0.0a 29.9 ± 0.1b 58.0 ± 1.2c 36.9 ± 3.6b

K 7.30 ± 0.2a 19.1 ± 0.1b 32.7 ± 0.7c 16.1 ± 1.6b

Cl 5.14 ± 0.0a 56.5 ± 0.1b 108 ± 2.4c 53.9 ± 5.3b

Mg 0.40 ± 0.0a 0.14 ± 0.1a 0.12 ± 0.0a -0.83 ± 0.2b

Ca -0.22 ± 0.3a -0.83 ± 0.8a 0.73 ± 0.2a -0.61 ± 0.2a

P 0.29 ± 0.1a 0.58 ± 0.1ab 0.74 ± 0.0b -0.46 ± 0.1c

abc values with the same letter in the same row are not significantly different (p < 0.05)

5.4 DISCUSSION

Sheep fed saltbush have less efficient rumen fermentation than sheep fed a control diet,

supporting my first hypothesis. Feeding saltbush increased the amount of methane

produced compared to sheep fed the no-salt diet (Table 5.2). The concentration of

volatile fatty acids in the rumen decreased, and the ratio of acetate to propionate in the

rumen increased. In addition to this, saltbush was less digestible than the no-salt pellet,

despite containing lower levels of fibre (Table 5.1).

Inefficient digestion means that sheep fed saltbush lose more energy and utilise less of

the available energy compared to sheep fed the control diet. This could help to explain

68

why sheep fed saltbush struggle to maintain weight. During the conversion of gross

energy to net energy (Figure 2.2), major energy losses occur in the faeces, urine and

methane production. A small amount of energy is also used in heat production. I did

not measure urine energy or heat production in this experiment, but sheep fed saltbush

lost significantly more energy in their faeces (Table 5.1) and as methane (Table 5.2)

compared to sheep fed the no-salt diet.

Loss of energy in the faeces is the largest and most variable loss of energy from feed,

and varies between 20 and 80% of gross energy intake (Standing Committee on

Agriculture 1990, Coleman and Henry 2002). The organic matter digestibility of

saltbush was only 48% compared to 66% for the no-salt diet (Table 5.1), and faecal

energy losses for saltbush were large.

The production of methane is another major inefficiency in ruminant production, and

the extra production of methane by sheep fed saltbush is an important consideration.

Up to 12% of digestible energy (Figure 2.2) can be lost as methane, and 10% is

considered the average value (Bryant 1965, Johnson and Johnson 1995, Ulyatt et al.

1997, Ulyatt et al. 2002). If we assume that 10% of digestible energy was lost from the

no-salt diet as methane, the 42% increase in methane production from the saltbush diet

compared to the no-salt diet (Table 5.2) means that the sheep fed saltbush were losing

14% of digestible energy as methane. Low voluntary feed intake of saltbush means that

there is unlikely to be a net increase in methane emissions from sheep grazing saltbush

in the field, but the increase in losses of digestible energy as methane is a major concern

for animal producers.

Of the remaining energy (metabolisable energy) available to the sheep, there were less

total volatile fatty acids produced per mL of rumen fluid in sheep fed saltbush compared

to the no-salt diet (Table 5.2). Volatile fatty acids are a major source of energy for

sheep, and normal rumen concentrations are between 70 and 130 mmol L-1 (Van Soest

1987, France and Siddons 1993). The concentration of volatile fatty acids in the rumen

fluid from sheep fed saltbush (55 mmol L-1) was below this level.

Sheep fed saltbush also had a higher ratio of acetate to propionate in their rumen fluid

(5:1) compared to the control diet, and the normal ratio of 3:1 (Table 5.2) (France and

Siddons 1993). The increase in the ratio of acetate to propionate can be linked to the

69

increase in methane production. Methanogens require hydrogen gas produced during

the synthesis of acetate from cellulose to make methane (Pelchen and Peters 1998), thus

more acetate means there is more substrate available for methane production. Similarly,

the bacteria that produce propionate compete with methanogens for hydrogen, so more

methane is correlated with less propionate. Not only do sheep fed saltbush lose more

faecal and methane energy compared to sheep fed a control diet but they have a lower

concentration of volatile fatty acids in their rumen fluid. Overall, this means that sheep

fed saltbush have less energy available for growth, maintenance and reproduction

compared to the sheep fed the control diet.

The decrease in rumen efficiency in sheep fed saltbush compared to the control diet was

not entirely due to the high level of salt in the feed, and my second hypothesis is

rejected. The high level of salt in saltbush was responsible for the decrease in total

volatile fatty acid concentration (Table 5.2), but was only partially responsible for the

decrease in organic matter digestibility (Table 5.1), and had no effect on methane

production, the concentration of methanogens in the rumen, or the ratio of acetate to

propionate in the rumen (Table 5.2).

One of the main consequences of feeding sheep a high salt diet, either salty pellets or

saltbush, is an increase in water intake (Meyer and Weir 1954). Hemsley et al (1975)

increased the water intake of sheep by adding salt to their feed (80 g day-1) and drinking

water (1% w/v). They did not measure a change in rumen volume, but reported a

decrease in ruminal volatile fatty acid concentration and the residence time of a

radioactive marker (Cr-EDTA) in the rumen. This was accompanied by an increase in

the amount of volatile fatty acids leaving the rumen unabsorbed. The reduced

concentration of volatile fatty acids in the rumen fluid of sheep fed salty diets in my

experiment is therefore likely to be due to the increased rate of removal of volatile fatty

acids from the rumen. The loss of volatile fatty acids from the rumen of sheep fed high-

salt diets would be exacerbated in the sheep fed saltbush as high ruminal pH inhibits

absorption of volatile fatty acids across the rumen wall (Table 5.2) (France and Siddons

1993). So although feeding sheep high salt diets may not cause a decrease in the total

amount of volatile fatty acids produced, sheep may be unable to utilise all of the energy

produced during rumen fermentation.

70

These results contradict those of Arieli et al (1989) who found a decrease in total

volatile fatty acid concentration in the rumen of sheep fed a mixed diet with added salt

compared to a control (no salt) diet, but not in the rumen of sheep fed saltbush (A.

barclayana). The authors suggested that the difference in volatile fatty acid

concentration between the salty diet and saltbush was due to the solubility of salts from

the ration. In the previous experiment (Chapter 4), I found that not all of the salt in

saltbush leaves was released in to the rumen. The presence of more soluble salts in the

salty diet compared to the saltbush would cause a greater increase in rumen osmolality

and potentially a greater subsequent increase in water intake. This would increase the

removal of volatile fatty acids from the rumen, decreasing ruminal volatile fatty acid

concentration. Based on the logic outlined above, the salty diet should have had a lower

rumen retention time than either the saltbush or control diets. However, Arieli et al

(1989) calculated the rumen retention time of the three diets to be 9.2, 12.4 and 16.7

hours for the saltbush, salt and control diets. The authors do not offer another

explanation for their results, and may have made an error in their measurements,

calculations or reporting of their results. Alternatively, the difference between my

results and those of Arieli et al (1989) could be due to the different types of saltbush

used (A. barclayana vs. A. nummularia).

The increase in water intake is probably also largely responsible for the decrease in the

organic matter digestibility of the saltbush and salty pellets compared to the control diet

(Table 5.1). This is despite the saltbush and salty pellets containing lower levels of

neutral detergent fibre and acid detergent fibre, which should make them more

digestible. Increased rumen dilution may reduce the colonisation of feed particles by

ruminal microorganisms and the increased rate of passage may flush feed particles from

the rumen before they can be fully digested, causing an increase in faecal energy losses

(Hemsley et al. 1975). However, the organic matter digestibility of the saltbush diet

was lower than the organic matter digestibility of the salty pellets despite containing

less salt (Table 5.1). This suggests that there are other factors involved in reducing the

organic matter digestibility and increasing faecal energy losses from saltbush. Saltbush

contains many secondary compounds, including oxalates, saponins, nitrates and tannins

(Norman et al. 2004) (Table 2.1). These compounds may reduce the organic matter

digestibility of saltbush by protecting feed particles from microbial digestion or

interfering with microbial metabolism (Van Soest 1987, Burritt and Provenza 2000,

Wallace 2004).

71

The presence of secondary compounds in saltbush is also likely to be responsible for the

increase in methane production by sheep fed saltbush (Table 5.2). Saltbush contains

high levels of oxalates (2-9 % DM) (Table 2.1), which are degraded by ruminal

microorganisms to produce carbon dioxide (Allison et al. 1977). Carbon dioxide and

hydrogen are the major substrates for methane production, and extra carbon dioxide

could be utilised by methanogens to produce extra methane. This would reduce the

amount of hydrogen available for propionate production (Table 5.2). Saltbush also

contains low levels of tannins (<0.1 % DM) (Table 2.1), which have been shown to

increase methane production in other experiments (Sliwinski et al. 2002, Waghorn et al.

2002).

The high methane production from the saltbush diets compared to the pellets is

particularly interesting because, based on the nutritive value of saltbush (Table 5.1),

methane production should actually have been lowest from the saltbush diet. Methane

production is positively correlated with the fibre content and digestibility of a ration

(Pelchen and Peters 1998, Chandramoni et al. 2000, Waghorn et al. 2002). The

saltbush diet contained the least amount of fibre and was the least digestible of the four

diets, so it would be expected to have the lowest methane production.

Waghorn et al (2002) suggested that methane emissions from ruminants could be

reduced by increasing the rate of passage of feed through the rumen. However, the

salty pellets in this experiment would have had a shorter rumen residence time

compared to the control pellets but there was no associated decrease in methane

production (Hemsley et al. 1975). Under practical conditions, decreasing methane

production by decreasing digestibility and increasing rate of passage may lead to an

increase in losses of faecal energy (Figure 2.2). Any benefit gained from reducing

methane production would be offset by a concurrent reduction in the amount of

digestible energy available to the sheep.

The increase in methane production by sheep fed saltbush was accompanied by a large

increase in the concentration of methanogens in the rumen fluid (Table 5.2). Because

there was a much larger increase in the concentration of methanogens compared to

methane, it can be assumed that the increase in methane production by sheep fed

saltbush is probably due to an increase in population size rather than activity. It is

72

possible that the methanogens that dominate the rumen of sheep fed saltbush produce

only small amounts of methane. Alternatively, the methane production could be

attributed to a small group of active methanogens.

It is worth noting here that high salt concentrations did not suppress methane production

or the number of methanogens in sheep fed the low-salt and high-salt diets, despite an

increase in rumen salinity (Table 5.2). In addition to increased rumen salinity, the

intake of high salt diets by sheep means that ruminal methanogens are also likely to face

an increase in the rate of passage and dilution of feed particles (Hemsley et al. 1975).

This indicates that ruminal methanogens are salt tolerant and can reproduce fast enough

to overcome the effects of the increased rate of passage.

Feeding both saltbush and formulated high-salt diets decreases the efficiency of rumen

fermentation, with consequences for ruminant production. Sheep fed salty diets lose

large amounts of energy in the faeces, and may utilise less of the energy available for

production (volatile fatty acids). Sheep fed saltbush face additional energy losses in

faecal and methane energy that cannot be explained by the high salt content of the

forage. These results may help to explain why sheep grazing saltbush struggle to

maintain weight, even when the nutritive value of the feed suggests it is adequate for

maintenance energy requirements.

An unexpected result from this experiment was the difference between saltbush and the

salty pellets in the apparent digestion and absorption of minerals. There was a smaller

proportion of Na, Cl, Mg, Ca and P digested in sheep fed saltbush compared to the salty

pellets, though there was no difference in K digestion (Table 5.3). In the first

experiment (Chapter 4), the rumen fluid from sheep fed saltbush had a lower electrical

conductivity (Table 4.2) than expected given the amount of salt in the diet, indicating

that not all the salt was released from the saltbush leaves. This also occurred in the

current experiment. My results demonstrate that some salt is retained in the saltbush

leaves throughout the digestive tract, as there were higher concentrations of minerals in

the faeces of sheep fed saltbush compared to the other diets (Table 5.3). This could be

caused by chemical binding of some elements to large, indigestible molecules, or the

physical enclosure of minerals in undigested fibre bundles. These molecules or bundles

manage to resist degradation not only by rumen microorganisms, but also by the more

acid conditions of the duodenal region. These results are consistent with those of

73

Playne et al (1978), who measured the rate of release of minerals from four different

plant materials. They found that some minerals were more resistant than others to

removal from plant material, with P being the most resistant, followed by Ca, Na and

Mg. K was by far the most easily removed mineral.

It is also interesting that there was a net loss of Mg, Ca and P from the sheep fed

saltbush (Table 5.3), and that this was not due to the large amount of salt in the diet.

Magnesium, Ca and P are stored in the skeleton, and may be mobilised when dietary

intake and absorption is inadequate (Underwood and Suttle 1999). The bone reserves of

Mg, Ca and P would be approximately 14, 590 and 288 g for the sheep used in this

experiment (Grace 1983). Sheep are unlikely to exhibit deficiencies in Ca or P for a

considerable period of time due to the size of their bone reserves. However, if the sheep

continued to lose 0.83 g Mg day-1 (Table 5.3), their bone reserves would be exhausted

in only 17 days. This is an important issue as sheep often graze saltbush for several

weeks at a time.

Magnesium is usually absorbed from the rumen and any excess is excreted via the

kidneys (Underwood and Suttle 1999). It is less soluble in rumen fluid with a high pH

(> 6) (Dalley et al. 1997b). The sheep fed saltbush had the highest ruminal pH in this

experiment (Table 5.2). Combined with the increased rate of passage of feed through

the rumen in sheep fed salty diets, the high rumen pH could account for decreased Mg

absorption. In addition to this, absorption of Mg from the entire gastrointestinal tract is

reduced when sheep are fed diets containing high levels of K (Newton et al. 1972,

Dalley et al. 1997a). Magnesium could also bind to P to form Mg phosphate, which is

insoluble (McDowell 1992). Magnesium deficiency causes hypomagnesaemic tetany

(grass tetany), where animals collapse and die within a few hours (Standing Committee

on Agriculture 1990, Brightling 1994). It is exacerbated by Ca deficiency.

Calcium deficiency results in loss of appetite, stunted growth and hypocalcaemia (milk

fever), which can cause death (Brightling 1994). Calcium is absorbed according to

need, so any excess is excreted via the faeces (Underwood and Suttle 1999). While we

cannot explain the net loss of Ca from sheep fed saltbush, there may be three

contributing factors to the decrease in absorption. First, the saltbush used in this

experiment had a high dietary cation-anion balance (Table 5.1). Diets with a dietary

cation-anion difference value greater than 100 are alkaline and have decreased Ca

74

absorption. Second, the Ca present in saltbush may be in the form of Ca-oxalates,

which are insoluble and not readily metabolised by rumen microorganisms (McDowell

1992, Underwood and Suttle 1999). And finally, all the sheep used in this experiment

were housed inside a shed and may not have been receiving enough vitamin D3, which

comes from sunlight. Vitamin D3 is essential to the efficient utilisation of Ca and P, and

this may help to explain why sheep on other diets did not absorb any Ca (Table 5.3).

My results indicate that saltbush may be unsuitable as a feed for sheep with high

nutritional demands, such as ewes during pregnancy and lactation. Some farmers in

Western Australia have reported unexplained ewe and lamb deaths on saltbush pastures,

although there have been no published studies to confirm this. It is important to

investigate the mineral balance of sheep fed saltbush in a field situation to avoid issues

related to Mg, Ca and P deficiency, such as milk fever/hypocalcaemia,

hypomagnesaemia and decreased milk yield.

75

Chapter 6:

What is the optimal level of barley to feed sheep grazing saltbush?

6.1 INTRODUCTION

In the previous experiment (Chapter Five) it was demonstrated that sheep fed saltbush

have less efficient rumen fermentation than sheep fed a control diet. This was

characterised by increased methane production, decreased total volatile fatty acid

concentration, an increase in the ratio of acetate to propionate, and reduced organic

matter digestibility. These inefficiencies are likely to be caused by a combination of the

high level of minerals (particularly NaCl and KCl) and secondary compounds in

saltbush foliage.

To improve the feeding value of saltbush pastures, producers often provide their sheep

with a supplement. Feeding straw improves the feed intake and weight gain of sheep

fed saltbush in the animal house but has not been successful in field situations (Warren

et al. 1990, Franklin-McEvoy et al. 2007, Norman et al. 2008). The provision of a

high-energy supplement, such as barley, is more promising.

Feeding barley as a supplement to a saltbush diet can improve the feeding value of

saltbush by providing energy to ruminal microorganisms to produce microbial protein,

stimulate carbohydrate digestion and detoxify secondary compounds (Hassan and

Abdel-Aziz 1979, Provenza et al. 2003). Feeding barley also reduces the water intake

of sheep fed saltbush, which decreases rumen dilution and the rate of passage of feed

particles through the rumen. However, the minimum amount of barley that is required

for sheep to maintain weight on saltbush has not been established. This is important

given the high cost of barley compared to saltbush in a commercial feeding system.

van der Baan et al (2004) found that feeding barley at 15% of the diet increased the

organic matter digestibility of saltbush (Figure 2.5). However, there was no further

improvement in digestibility when sheep were fed 30 or 45% barley. This indicates that

there may be an optimal level of supplementation for sheep grazing saltbush. That is,

there will be a minimal amount of barley required to improve the efficiency of rumen

76

fermentation in sheep grazing saltbush, but also a level beyond which no additional

benefit is obtained.

I hypothesised that there is an optimal amount of barley required to improve the

efficiency of rumen fermentation (increased organic matter digestibility, increased

volatile fatty acid concentration, decreased acetate: propionate, and decreased methane

production) in sheep fed saltbush.

6.2 MATERIALS AND METHODS

6.2.1 Experimental design

Thirty sheep with an average weight of 42.7 ± 0.4 kg were individually penned in the

animal house for five weeks. Sheep were fed the control diet (no-salt, Table 3.1) for

two weeks and were then allocated to one of six experimental diets based on liveweight

(Table 6.1). All the sheep were fed saltbush (A. nummularia) plus straw and barley at

maintenance for three weeks and the efficiency of rumen fermentation was measured.

Liveweight was measured before feeding once every week.

6.2.2 Diets

Due to the risk of sheep developing acidosis when fed barley alone, equal amounts of

straw and barley (DM weight basis) were substituted for saltbush at 0, 20, 40, 60, 80

and 100% of the maintenance diet (Table 6.1 and 6.2). The straw and barley were

combined in a pellet. This was then mixed with the saltbush by hand.

77

Table 6.1 Composition and allocation of treatment diets. SB: 100% saltbush, BS20:

20% barley and straw, BS40: 40% barley and straw, BS60: 60% barley and straw,

BS80: 80% barley and straw, BS: 100% barley and straw.

% of maintenance diet Diet

Saltbush Barley & straw Number of animals

SB 100 0 7

BS20 80 20 7

BS40 60 40 5

BS60 40 60 4

BS80 20 80 3

BS100 0 100 3

Table 6.2 Nutritive value of the experimental diets. SB: 100% saltbush, BS20: 20%

barley and straw, BS40: 40% barley and straw, BS60: 60% barley and straw, BS80:

80% barley and straw, BS: 100% barley and straw pellet.

Diets Feed component (% DM) SB BS20 BS40 BS60 BS80 BS100

Na 8.83 7.13 5.43 3.73 2.03 0.34

K 2.00 1.86 1.72 1.59 1.45 1.32

Cl 14.1 11.5 8.83 6.18 3.53 0.87

Ash 35.9 29.6 23.4 17.6 12.1 6.00

N 2.41 2.17 1.93 1.70 1.46 1.22

Neutral detergent fibre 23.5 28.3 36.3 41.6 48.0 64.9

Acid detergent fibre 12.5 15.2 16.0 22.3 24.6 25.4

Because the aim of this experiment was to describe the pattern of the relationship

between the level of barley fed and the efficiency of fermentation, equal numbers of

sheep were not allocated to each treatment diet. More animals were allocated to the

diets containing more saltbush (Table 6.1) as we expected there to be more variation in

78

the parameters measured. One animal had to be removed from the experiment due to

poor appetite and weight-gain, so only 29 animals were used.

6.2.3 Digestibility

All sheep were fitted with faecal harnesses for the final ten days of the experiment.

Faecal samples were collected for the final seven days for measurement of organic

matter digestibility (see section 3.3.2).

6.2.4 Rumen pH, salinity and volatile fatty acid concentration

Around 50 mL of rumen fluid was taken from all animals three hours after feeding on

the final day of the experiment. The pH and electrical conductivity of rumen fluid were

measured immediately, and the remaining rumen fluid was divided up for measurement

of volatile fatty acids and in vitro methane production.

6.2.5 Methane production

Methane production was measured in vitro during this experiment. The measurement

of methane production in vivo in Chapter Five of this thesis supported the results

previously obtained in vitro, so this provided confidence that the in vitro procedure

would accurately reflect what would happen in vivo (Mayberry 2003). Measuring

methane production in vitro instead of in vivo meant that the sheep were confined to the

animal house for a shorter period of time.

The ability of microbial populations in rumen fluid to produce methane was measured

using the method described by Zinder (1998). The substrates provided to the

methanogens were sodium formate, sodium acetate, hydrogen and carbon dioxide.

On the day prior to rumen fluid collection, 100 µL of 12.5% sodium acetate and 100 µL

of 12.5% sodium formate were aliquotted into 20 mL bellco tubes under anaerobic

conditions. The tubes were sealed, crimped and autoclaved at 121˚C for 20 minutes.

79

On the day of sampling, 5 mL of rumen fluid was dispensed into each tube using 18 G

needles. Three tubes were used per sheep. The tubes were then overpressurised to 2

bar using a hydrogen and carbon dioxide gas mix (80/20) and incubated with shaking at

39˚C.

After six hours the pressure of gas in the headspace was measured using a digital

pressure meter (Greisinger electronic, Germany). Fermentation was stopped by adding

0.2 mL formalin, and tubes were refrigerated before measurement of methane

production.

The concentration of methane in the headspace above the rumen fluid mixture was

measured using gas chromatography (Agilent 6890 series GC system, USA). The

amount of methane produced was calculated using the Ideal Gas Law;

PV = nRT

Where P: headspace pressure (atm)

V: headspace volume (L)

n: amount of methane (moles)

R: ideal gas constant

T: temperature (K)

6.3 RESULTS

6.3.1 Rumen pH and salinity

The salinity and pH of rumen fluid tended to decrease as more of the barley and straw

pellet was included in the diet (Figure 6.1a & b). There was no further decrease in

rumen pH when more than 60% barley and straw was included in the diet.

80

6.3.2 Digestibility

The organic matter digestibility of the ration increased when more of the barley and

straw pellet was included in the diet (Figure 6.1c). There was no further improvement

in digestibility when more than 60% barley and straw was included in the diet.

6.3.3 Methane production

The amount of methane produced from rumen fluid in vitro decreased when more of the

barley and straw pellet was included in the diet (Figure 6.1d). The average amount of

methane produced by sheep fed the diet containing 80% barley and straw was lower

than the average methane production for all the other diets, and did not fit the curve

fitted to the data. If this is discarded, there was no further reduction in methane

production when sheep were fed more than 60% barley and straw.

6.3.4 Volatile fatty acid concentration

The concentration of total volatile fatty acids in the rumen fluid increased when more of

the barley and straw pellet was included in the diet (Figure 6.1e). The exceptions to this

were the sheep fed 80% barley and straw. Total volatile fatty acid production continued

to increase when more than 60% barley and straw was included in the diet.

The ratio of acetate to propionate decreased when sheep were fed diets containing more

barley and straw, with the exception of the diet containing 80% barley and straw

(Figure 6.1f). If this group is not included, the ratio of acetate to propionate tends to

stabilise when 60% barley and straw is fed.

81

Electrical conductivity (mS cm-1)

0

10

20

30

0 20 40 60 80 100

% barley & straw

(a) Rumen salinity

pH

0

2

4

6

8

0 20 40 60 80 100

% barley & straw

(b) Rumen pH

Organic matter digestibility (%)

% barley & straw

(c) Organic matter digestibility

Methane (mmoles)

0.00

0.01

0.02

0.03

0.04

0 20 40 60 80 100

% barley & straw

(d) In vitro methane production

Volatile fatty acids (mmoles -1)

0

20

40

60

80

100

0 20 40 60 80 100

% barley & straw

(e) Total volatile fatty acid concentration

Acetate:propionate

0

2

4

6

8

0 20 40 60 80 100

% barley & straw

(f) Ratio of acetate: propionate

Figures 6.1 Ruminal environment and efficiency of rumen fermentation in sheep fed

saltbush and offered different proportions of a barley and straw pellet. The broken line

in figures 6.1d, e and f is fitted to all the data. The solid line in figures 6.1d, e and f

excludes sheep fed 80% barley and straw.

0

20

40

60

80

0 20 40 60 80 100

82

6.4 DISCUSSION

Substituting barley and straw for saltbush improved the efficiency of rumen

fermentation. The digestibility of the diet and concentration of volatile fatty acids in

rumen fluid were increased, while in vitro fermentation of methane and the ratio of

acetate to propionate in the rumen fluid were reduced. The efficiency of rumen

fermentation by sheep fed saltbush plus the barley and straw pellet equalled that of the

sheep fed 100% barley and straw at the inclusion of 60% barley and straw in the diet.

This supports the hypothesis that there is an optimal amount of barley required to

improve the efficiency of rumen fermentation in sheep fed saltbush.

Saltbush contains high levels of nitrogen, but a large proportion of this is non-protein

nitrogen (Benjamin et al. 1992, Masters et al. 2001). There is insufficient energy in

saltbush for ruminal microorganisms to convert dietary nitrogen into microbial protein,

and sheep fed saltbush without a supplement may have a negative nitrogen balance.

Hassan and Abdel-Aziz (1979) fed sheep saltbush (A. nummularia) plus 0, 50, 100 or

150 g barley day-1, and reported that sheep fed 0 or 50 g barley-1 had a negative nitrogen

balance, but those fed 100 or 150 g barley day-1 were able to retain nitrogen. This was

due to an increase in the amount of dietary nitrogen digested. The provision of barley

grain or a barley and straw pellet to sheep fed saltbush provides a source of readily-

available energy to ruminal microorganisms. Microorganisms can use this extra energy

for growth and to convert dietary protein from the saltbush leaves into ammonia and

then microbial protein, stimulating carbohydrate digestion, and consequently increasing

organic matter digestibility and the concentration of volatile fatty acids in the rumen

(Figure 6.1c & e) (Annison et al. 2002).

Based on the partial digestibility of the saltbush (44.6%) and barley and straw (64.2%)

diets, I expected the organic matter digestibility of the diets containing 20, 40, 60 and

80% barley and straw to be 48.5, 52.4, 56.3 and 60.3% respectively. Instead, the

organic matter digestibility of these diets was three to seven percent higher, at 51.2,

57.3, 63.2 and 64.4% (Figure 6.1c). This increased digestibility means that sheep will

gain more energy per unit of saltbush consumed when they are provided with a barley

and straw supplement.

83

Sheep fed saltbush plus barley grain can also consume more saltbush than

unsupplemented sheep. Weston (1988) found that the increased digestibility of a

roughage diet (lucerne and wheaten hay) caused by providing a cereal grain supplement

meant that sheep fed the supplemented diet ate 5% more feed and spent 24% less time

eating and 14% less time ruminating than sheep fed the roughage diet alone. Hassan

and Abdel-Aziz (1979) measured an increase in the voluntary intake of saltbush and an

improvement in liveweight gain when sheep were offered 150 g barley day-1, thus an

improvement in rumen fermentation can translate to an improvement in animal

production. An increase in the amount of saltbush consumed by sheep was not

measured in this experiment because the amount of feed on offer to the animals was

restricted to a maintenance ration.

My results support those of Hassan and Abdel-Aziz (1979), who reported an increase in

the digestion of crude fibre and protein by sheep fed saltbush (A. nummularia) ad

libitum when they were provided with a supplement of 100 or 150 g barley day-1. The

authors also reported a decrease in water intake by sheep. While I did not measure

water intake, I did record a decrease in ruminal salinity when the barley and straw pellet

was substituted for saltbush (Figure 6.1a). A decrease in ruminal salinity would

decrease the amount of water consumed by the sheep (Carter and Grovum 1990). A

reduction in water intake would reduce the dilution of volatile fatty acids in the rumen

fluid (Figure 6.1e) and the rate of passage of feed particles through the rumen.

Increased residence time of feed particles in the rumen would allow the ruminal

microorganisms more time to digest the feed, increasing organic matter digestibility

(Figure 6.1c).

The provision of a high-energy substrate to ruminal microorganisms may also improve

the fermentation of saltbush by allowing sheep to ingest more toxins in their diet.

Energy and protein are required for the detoxification and elimination of toxic

compounds, including oxalates, saponins, tannins and nitrates, which are found in

saltbush (Provenza et al. 2003, Norman et al. 2004). In the previous experiment

(Chapter Five), sheep fed saltbush produced more methane and had a higher

concentration of methane-producing archaea in their rumen than sheep fed a control

diet. It was concluded that the increase in methane fermentation was possibly due to the

presence of secondary compounds (oxalates or tannins) in the feed. Detoxification of

84

these compounds, facilitated by the energy available from the barley and straw pellet,

may account for the decrease in methane production observed in this experiment

(Figure 6.1d).

The provision of energy in the form of a barley and straw pellet improves the efficiency

of rumen fermentation (organic matter digestibility, methane production, ratio of acetate

to propionate) until energy is no longer the limiting factor in rumen fermentation

(Figure 6.1c, d & f). If sufficient energy is provided for formation of microbial protein,

breakdown of carbohydrates and detoxification and elimination of secondary

compounds at the inclusion of 60% barley and straw, then the inclusion of 80 or 100%

barley and straw in the diet will not lead to further improvements in rumen

fermentation.

When more than 60% barley and straw is included in the diet, ruminal pH becomes the

limiting factor in rumen fermentation (Figure 6.1b). Barley is rich in simple

carbohydrates that are rapidly broken down by ruminal microorganisms, causing

ruminal pH to drop (Mackie et al. 2002, McDonald et al. 2002). Low ruminal pH limits

the rate and extent of fibre digestion by inhibiting the activity of cellulose-digesting

microorganisms (Van Soest 1987, McDonald et al. 2002). Weston (1988) found that

the infusion of buffer salts (sodium and potassium bicarbonate) into the rumen of sheep

fed roughage and a cereal supplement prevented a decrease in ruminal pH and improved

fibre digestion. While this is not practical in a field situation, buffering compounds

could be mixed with barley grain before it is fed to sheep grazing saltbush.

Rumen methanogens are also sensitive to low ruminal pH and the reduction in ruminal

pH when the barley and straw pellet was substituted for saltbush could account for the

decrease in methane fermentation (Figure 6.1d) (Van Kessel and Russell 1996).

Based on the improvement in the efficiency of rumen fermentation, the optimal level of

inclusion of barley and straw was 60% of the diet (Figure 6.1c, d & f). This is much

higher than the value of 15% reported by van der Baan et al (2004), and the difference

between the two experiments is probably due to the type of supplement used. Van der

Bann et al (2004) fed sheep straight barley grain, but in this experiment the barley grain

was combined with straw in a pellet because of the risk of sheep developing acidosis.

Norman et al (2008) found that the provision of roughage supplements (hay or straw) to

85

sheep grazing saltbush had no effect on animal performance, despite an increase in

energy intake. This is likely to be due to the digestibility of the roughage supplements

(Thomas et al. 2007a). van der Baan et al (2004) did not report the organic matter

digestibility of the barley used in their experiment, but it is likely to be higher than the

organic matter digestibility of barley combined with straw. The high organic matter

digestibility of the barley grain means that sufficient levels of energy for formation of

microbial protein, breakdown of carbohydrates, and detoxification and elimination of

secondary compounds would be available at much lower levels of substitution. The pH

of rumen fluid would also decrease more rapidly, limiting fermentation sooner.

In this experiment, barley grain was combined with straw to prevent sheep from

developing rumen acidosis, however, this may also be a more realistic scenario than

feeding sheep straight saltbush and barley grain. In the field, saltbush stands consist of

saltbushes plus a volunteer or sown understorey of annual grasses and legumes (Figure

2.1). This means that sheep grazing saltbush also have access to a supply of roughage

of similar quality to the straw used in this experiment. Fancote (2007) measured the

proportion of saltbush and understorey in the diet of sheep grazing saltbush during late

autumn and reported that the understorey comprised an average of 54% of the diet

selected by sheep.

If the digestibility of the diet is expressed in terms of how much barley was included in

the ration, instead of barley and straw, my results can be directly compared to those of

van der Baan et al. (2004). When the results from both experiments are combined

(Figure 6.2), it appears that the optimal level of supplementation is to feed barley grain

at approximately 20% of the maintenance diet.

86

Figure 6.2 Organic matter digestibility (OMD) of saltbush based diets containing

different proportions of barley grain. Results are from this experiment and that of van

der Baan et al. (2004).

The concentration of volatile fatty acids in the rumen fluid continued to increase after

the inclusion of 60% barley and straw in the diet (Figure 6.1e) as the extra energy in the

diet was converted directly into volatile fatty acids. In addition to this, the low ruminal

pH caused by rapid fermentation of the barley would increase absorption of volatile

fatty acids across the rumen wall (France and Siddons 1993). This means that even

though feeding more than 60% barley and straw does not improve the efficiency of

rumen fermentation, more energy will be available to the sheep for growth and

production. In this case it is up to the producer to weigh up the cost of the barley grain

supplement against the improvement in animal production.

The results from this experiment that are difficult to explain are the methane and

volatile fatty acid production in rumen fluid from the group of sheep fed 80% barley

and straw. The measurements from these sheep taken from the rumen samples

(methane, total volatile fatty acid concentration, acetate to propionate ratio) consistently

fell outside the relationship demonstrated at all other levels of supplementation (Figure

6.1d, e & f). In contrast to this, the results for organic matter digestibility (Figure 6.1c),

which were an average of measurements taken over several days, do not deviate from

the relationship. Because of this, I believe that there was an error in the sampling or

analysis of samples taken from these sheep. This idea is supported by the low methane

0

20

40

60

80

0 10 20 30 40 50

% barley in diet

OMD (%)

this experiment

van der Baan et al. (2004)

87

production and high acetate to propionate ratio for this group (Figure 6.1d & f). A

decrease in methane production should be accompanied by a decrease in the ratio of

acetate to propionate in the rumen fluid (Van Soest 1987, France and Siddons 1993), so

these results cannot be correct. While I cannot explain why these results deviate from

the relationship demonstrated at all other levels of supplementation, it is worth noting

that the inclusion of these results in the curve did not have a major effect on the

relationship between the level of supplementation and the efficiency of rumen

fermentation or the conclusions I have drawn from this data (Figure 6.1d, e & f).

The results from this experiment have demonstrated that there is an optimal amount of

barley to improve the efficiency of rumen fermentation in sheep fed saltbush. In this

experiment, the optimal level of barley and straw to feed sheep eating saltbush in the

animal house was 60% of the maintenance diet. However, there are several differences

between experiments run in the animal house and what happens in the field. In this

experiment, the sheep were fed at maintenance, and the proportions of saltbush and the

barley and straw pellet available were decided by me. In a field situation, sheep have

access to saltbush ad libitum, and are able to choose how much saltbush, understorey

and barley they consume. They also need to compete with other members of the flock

for feed, so some sheep will consume more barley than others. In addition to this, it is

impractical for farmers to feed out barley every day and it is likely that several days’

worth of barley will be provided to the whole flock at once. Further research is

therefore required to determine the optimal level of supplementation for sheep grazing

saltbush in the field and how the supplementation is managed.

88

Chapter 7:

General discussion

Sheep fed saltbush have inefficient rumen fermentation compared to sheep fed a control

diet. This could explain why sheep grazing saltbush struggle to maintain weight,

regardless of how much saltbush they consume. I hypothesised that the poor production

of sheep grazing saltbush pastures would be due to the high level of NaCl and KCl in

the saltbush leaves. However, sheep fed a formulated ration containing equally high

levels of NaCl and KCl did not experience the same effects on rumen fermentation,

despite an increase in ruminal salinity.

This does not mean that diets containing high levels of salt are without their problems.

Feeding pellets containing high levels of NaCl and KCl caused a large increase in the

salinity of the rumen fluid (Chapter Four). Although not measured in this thesis, it is

known that increasing the salinity of rumen fluid causes an increase in water intake

(Carter and Grovum 1990). The subsequent increase in rumen dilution and outflow

causes a decrease in organic matter digestibility, and the concentration and absorption

of volatile fatty acids in the rumen, reducing the amount of metabolisable energy

available to the sheep (Chapter Five). Combined with low feed intakes, this means

high-salt diets are far from an ideal feed.

In addition to the energy losses associated with high salt intakes, sheep grazing saltbush

also lose energy in the production of excess methane (Chapter Five). This is probably

due to the presence of secondary compounds (oxalates or low levels of tannins) in the

saltbush leaves, as increasing the amount of salt in the pelleted diets had no effect on

methane production (Sliwinski et al. 2002). Sheep fed saltbush also lose more energy

through the faeces than sheep fed a formulated high-salt diet. Given these additional

energy losses, it is hardly surprising that sheep on saltbush pastures struggle to eat

enough saltbush to maintain weight.

One of the most interesting results from this thesis was the accumulation of K in the

rumen of sheep fed saltbush and the formulated high salt diets (Chapter Four).

Absorption of Na and K usually occurs in the small intestine. Given the increased flow

rate of liquid and small particles through the rumen, I expected Na and K to be removed

89

from the rumen at a similar rate. However, while the concentration of Na in the rumen

appeared to be well-regulated, and returned to the pre-feeding level at the end of the

day, there was significantly more K in the rumen fluid of sheep fed high salt diets

compared to the control diet at all times. The importance of the rumen for absorption of

Na may increase in sheep fed high-salt diets. There was a significant increase in the

apparent absorption of K throughout the entire digestive tract in sheep fed the high salt

diets compared to the control diet, so K must continue to be absorbed from the small

intestines.

An unexpected but important result from this thesis was the net loss of Mg, Ca and P

from sheep fed saltbush (Chapter Five). It appears that these minerals are being

mobilised from skeletal reserves, and sheep may be at risk of developing mineral

deficiencies, which can reduce animal production and cause stock deaths. In addition to

possible Mg, Ca and P deficiencies, Masters et al (2007) have warned about the risk of

sheep grazing saltbush developing Cu deficiencies as a result of high sulphur intakes.

Given that saltbush is sometimes grazed by sheep with high nutritional demands, such

as pregnant or lactating ewes, it is important to further investigate the mineral balance

of sheep grazing saltbush.

Because saltbush is one of the only plants able to grow and produce green feed for

sheep on salt-affected land, it is important that researchers provide management options

for farmers to maximise sheep performance. Feeding barley as a supplement improves

the efficiency of rumen fermentation in sheep fed saltbush by providing extra energy to

ruminal microbes to produce microbial protein, stimulate carbohydrate digestion and

detoxify secondary compounds. In my experiment the efficiency of digestion of a

saltbush-based diet was equal to that of a barley and straw pellet after the inclusion of

60% barley and straw in the ration (Chapter Six). Lower levels of supplement are likely

to be required if straight barley grain is fed to sheep fed saltbush.

An alternative (or complement) to feeding barley supplements would be to provide

sheep with a mixture of plant species. Saltbush can have localised effects on soil

salinity by removing salt from the soil and lowering saline water tables (Barrett-

Lennard and Galloway 1996), allowing less salt-tolerant plants to be sown between the

saltbush shrubs. Pastures could also be sown on adjacent, non-salty sites. Thomas et al

(2007a), reported that sheep will select combinations of low and high salt diets to meet

90

their nutritional requirements. The provision of low-salt alternatives to saltbush could

help to minimise the negative effects of saltbush on rumen fermentation by decreasing

rumen salinity, water intake and rumen outflow. Plants with different types and

combinations of secondary compounds could also be included in the mixture to help

reduce the amount of energy lost during methane production. For example, the legumes

Lotus corniculatus and Hedysarum coronarium (sulla) contain high levels of condensed

tannins, and can reduce methane emissions from sheep (Waghorn et al. 2002, Ramirez-

Restrepo and Barry 2005).

The results from this thesis demonstrate that inefficient rumen fermentation in sheep fed

saltbush could contribute to poor animal performance. Diets containing high levels of

NaCl and KCl provide low levels of net energy to sheep, but sheep grazing saltbush

face additional energy losses in faecal and methane energy. Feeding high-energy

supplements such as barley can improve the efficiency of rumen fermentation in sheep

fed saltbush and help sheep to maintain weight. I have demonstrated that in the animal

house there is an optimal level of barley and straw to feed sheep grazing saltbush, based

on the improvement in the efficiency of rumen fermentation. If this is also true for field

situations, and correlates to an improvement in sheep performance, researchers can

implement a cost-effective supplementation strategy for farmers who graze their sheep

on saltbush pastures, resulting in more productive and profitable grazing systems for

salt-affected land.

91

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